CO6-1

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

N. Miyoshi and T. Takahashi1
Department of Tumor Pathology, Faculty of Medicine,
University of Fukui, and1
Research Reactor Institute, Kyoto University
INTRODUCTION: The LINAC (linear particle accelerator) technology in the milli and tera-hertz 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 spectra of a sectioned tissue of raw brain tumor C6 model as a collaborate study. The absorption spectra in the tera-hertz 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 was estimated a longer relaxation time component of the induced electricity forwater molecules [2-3] in the raw tumor tissue.
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 this study.
EXPERIMENTS: (1) Instrument of Near-field intera-hertz region: The photograph of the instrument
was shown in Fig. 1. Mark-A: Pre-probe Wiston
cone; 50-10mm diameter, Length=60mm; the irradiate
diame-ter=0.775mm; The concentrate light probe(diame-ter)=3mm. The instrument was developed by
Dr. T. Takahashi in KURRI. [Fig. 1]

A(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) in Figures 2-3 or with 2 plates of cover glasses(thickness=130-170 m) in Figure 4, respectively underfreezing condition (-20 C) before the measurements.
RESULTS: Two different penetration materials of saran-wrap film and cover glasses were mapping measuredof spectra of two different C6 tumor model tissues asshown in Figures 2-3 and Figure 4, respectively.

[Fig. 3] H.& E.-stained Images and linear mapping
line and the absorbance at 9.69 cm-1

[Fig. 4] C6 glial tumor model tissue, theH.&E.-stained Image, the transmit spectra, and thelinear mapping analysis of the different depth areas-I

From these 2 linear mapping data (Figures-3, and 4),
there were presented higher absorbance components at
7-9 cm-1 in necrotic cancer region in these linear analysis
from Figures 3 and 4 even the penetrate materials weredifferent. It was estimated that the specific water molecules at 7-9 cm-1 region might be presented in the necrotic cancer area which the large amount of lipid components induced in the necrotic cancer area. These environment condition will control the water molecule conformation (looks like a free water which the hydrogenbond will be longer, 0.295 nm than that, 0.273 nm of thebinding water molecules in viable cancer area) around thelipid components in the cancer region. These estimation
will be more needed to be reappear the spectra, especially,
the diffraction effect in these spectra data to remove fromthe data.
REFERENCES:[Fig. 2] H.&E.-stained Images and linear mapping
line and the transmitted spectra[1] P. C. Ashuworth, et. al., Optics Express, 17(14) (2009)
12444-12454.
[2] T. Fukasawa, et al., Phys. Rev. Let., 95 (2005) 197802.
[3] Hiroyuki Yada, et al., Chem. Phys. Let., 464 (2008) 166-170 .

採択課題番号24002 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、Andriana B.Bibin（京大・原子炉）高橋俊晴

共同通常

CO6-2

Carotenoid-Producing Lactobacillus plantarum Is Resistantto -Ray Irradiation

K. Oda, T. Saito1, Y. Matoba, Y. Kubono, M. Noda,
T. Kumagai and M. Sugiyama
Department of Molecular Microbiology and Biotechnology, Graduate School of Biomedical and Health Sciences,
Hiroshima University
1
Research Reactor Institute, Kyoto University

INTRODUCTION: Carotenoids are a group of coloredterpenoids with antioxidant properties. Especially, carotenoids have a unique radical scavenging and singlet oxygen quenching activities [1]. Therefore, carotenoids may
prevent the organism from -ray irradiation through its
scavenging and quenching activities. Lactic acid bacteria(LAB) are Gram-positive, low-GC, microaerophilic, rod
or cocci that ferment sugar to produce lactic acid, andhave been used to ferment foods for at least 4000 years.
Carotenoid-producing LAB may have an industriallyworth for producing the fermented food with antioxidant
effect. Lb. plantarum is one of LAB species used as probiotic microorganism for many fermented foods. In thisstudy, we investigated whether carotenoid-producing Lb.
plantarum strains are resistant to -ray irradiation.
EXPERIMENTS: Lb. plantarum strains used in thisstudy are described in Table 1. For their culturing, MRS
broth (Merck) were used. Each strain was grown in MRS
broth at 30˚C for 24 h, and the resulting broth contained
about 2×109 cells per ml. Before irradiation, cells were
washed with PBS buffer and resuspended into the same
buffer. Cell suspensions were irradiated with -ray from a60
Co source at a dose rate of 717 Gy per h. To measure
the number of surviving bacteria after irradiation, irradiated cell cultures were diluted appropriately, and thenplated on MRS agar in triplicate. Plates were incubated at
30˚C and colonies were counted after 72 h of incubation.
In Fig. 1, log of the ratio of the number after the treatment with irradiation to that without irradiation is plottedagainst the total dose.
RESULTS AND DISCUSSION: As shown in Fig. 1,
-ray irradiation experiments indicated that D10 of two
carotenoid-producing strains against the -ray irradiation(470 and 430 Gy for 3930 and SN35N strains, respectively) was higher than that of carotenoid-nonproducing
strain (190 Gy for SN13T strain). In addition, SN13T ismore sensitive toward the exposure of hydrogen peroxide,
one of the active oxygen, than 3930 and SN35N (Table 1).
These results suggested that carotenoid produced by LAB
is involved in the protection from active oxygen generated by -ray irradiation and from hydrogen peroxide. On
the other hand, whereas 3930 and SN35N show almost

the same resistance toward -ray irradiation, MIC towardhydrogen peroxide of 3930 (8 mM) was higher than thatof SN35N (6 mM). SN35N produces a plenty of extracellular polysaccharide, but 3930 and SN13T do not. The
extracellular polysaccharides produced may be involvedin the resistance toward hydrogen peroxide in the SN35N
strain.
Table 1.
Lactobacillus plantarum strains
Carotenoid Extracellular
MIC toward
Strainproducpolysaccharide H O c (mM)
2 2
tiona
productionb
3930
+
8
SN35N
+
+
6
SN13T
4
a
Judged by color of the cell pellets obtained after centrifugation: +, yellow; -, white.
b
Judged by turbidity of culture supernatant after centrifugation: +, cloudy; -, clear.
c
Hydrogen peroxide resistance assay were carried using
overnight cultures grown at 30˚C. Cells were washedwith PBS buffer and resuspended into the same buffer.
1×104 cells per spot was loaded on MRS agar containing
indicated concentration (4, 6, and 8 mM) of hydrogen
peroxide, which was prepared at the time of use. Plates
were incubated at 30˚C for 72 h. MIC, minimum inhibitory concentration.

Fig. 1. Response of Lb. plantarum strains to -ray irradiation. Strains were irradiated with the indicated doseof -ray and the survival ratio was measured as described above. ◆, 3930; ▲, SN35N; ■, SN13T. D10
represents the dose of irradiation causing death for 90 %of total cells.
REFERENCES:[1] A. Clauditz et al., Infect Immun., 74 (2006)
4950-4953.

カロテノイド産生植物乳酸菌を用いた
共同通常
低線量放射線被曝低減技術の開発（広大院・医歯薬保健学）小田康祐、的場康幸、宇田成利、熊谷孝則 （京大・原子炉）齊藤毅
採択課題番号 24003

CO6-3

Estimation of Damage Localization in DNA Irradiatedwith Ionizing Radiations in Water

K. Akamatsu, N. Shikazono and T. Saito1
Irradiated Cell Analysis Research Group, Quantum Beam
Science Directorate, Japan Atomic Energy Agency
1
Research Reactor Institute, Kyoto University

INTRODUCTION: It is known that DNA lesions induced by ionizing radiation and chemicals can cause
mutation and carcinogenesis. In particular, ‘clustereddamage’ site, that is a DNA region with multiple lesions
within one or two helical turns, is believed to be hardly
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 degree of localization of
apurinic/apyrimidinic(AP) sites on 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]. Now we have tried to apply the method to
4
He2+- and 60Co -irradiated DNA.
EXPERIMENTS:●Sample preparation and He beam irradiation
Plasmid DNA digested by Sma I was used (linear
formed). One hundred microliters of the DNA aq. (0.5g/L) was transferred to an irradiation chamber (Fig.1),
and was irradiated with linear energy transfer (LET) of ~70 keV/m of 4He2+ beam (TIARA, Japan Atomic Energy Agency), which was controlled by a depth-tunable cellirradiation equipment at r.t.. 60Co -rays (Kyoto University Research Reactor Institute: KURRI) were also used
as a standard radiation source.

Fig.1. Irradiation chamber (mm)●Preparation of fluorophore-labeled irradiated DNA andthe fluorescence spectroscopy for 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 37°C. The fluorophore-labeled DNA
was purified by ethanol precipitation. Twenty microlitersof 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 themeasurement, the enzyme cocktail containing DNase I

and phosphodiesterase I was added to the solution, and it
was incubated for 2 h at 37°C. E values were calculated
from the donor intensity before/after the digestion.
RESULTS AND DISCUSSION:

Fig.2. Relationship between absorbed dose and AP density for
He ion beam (■) and 60Co -rays (●).

Fig.3. Relationship between AP density and FRET efficiency
for He ion beam (■) and 60Co -rays (●). The dashed lines
indicate theoretical lines for randomly-distributed AP in DNA.

The He beam is stopped completely by DNA aq. (1mm depth, see Fig.1), and all of the energy is transferred into the solution. It should be noted that the energy deposition may not be homogeneous although diffusion of DNA molecules can be promoted by vibrationof the chamber during irradiation. Fig. 2 shows clear
difference in the AP density (the number of AP sites
produced/total DNA base pairs in the solution) between
these radiation sources. Elimination of OH radicals byrecombination would be responsible for the low level
AP production for the high-LET He beam. In Fig.3, decrease of E for 60Co -rays would be caused by grossfragmentation of DNA due to the numerous lesions.
More data of lower dose region are needed. The E values for He beam are much higher than those for randomly-distributed AP. This seems that He beam produces clustered AP regions more frequently than random case. However, deeper consideration in heterogeneity of the energy deposition should be important for
more proper estimation of the damage localization. Improvement of the irradiation system may also be needed.
REFERENCE:[1] K, Akamatsu, N. Shikazono, Anal. Biochem. 433(2013) 171-180.

採択課題番号 24005 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）齋藤 毅

共同通常

CO6-4Structure Specificity of Radio-Induced Biomolecule Damage andIts Effect on Radio-Biological Consequence

H. Terato and T. Saito1
Analytical Research Center for Experimental Sciences,
Saga University
1
Research Reactor Institute, Kyoto University
INTRODUCTION: We have thought existence of a
specific form of radiation damage, and are recently starting to consider clustered DNA damage (CDD) as one ofthe damage. CDD contains multiple lesions in the limited
region of target DNA molecule caused by passage ofradiation beam track. High-LET radiations such as heavy
ion beams generate more condense and vast form of the
track than low-LET radiation such as gamma-ray and
X-rays. Thus, heavy ion beams are thought to produce
larger number of CDD than low-LET radiations. In thisregard, we indicated that yields of CDD decreased inelevation of the LET in the DNA molecule target irradiated in vitro (gamma > carbon > iron) [1]. We also reported that the isoltated DNA damage showed similar
trend for the LET’s elevation. In this study, we simultaneously analyze the yields of DNA damage including
CDD and surviving fractions of the irradiated cells to
discuss the connection between the CDD and the impact
of radio-biological effectiveness in heavy ion beam irradiation.
EXPERIMENTS: Chinese hamster ovary (CHO) AA8
cells growing exponentially were irradiated by
gamma-ray (0.2 keV/µm), and carbon (13 keV/µm), silicon (55 keV/µm), argon (90 keV/µm) and iron (200keV/µm) particle beams, respectively (parenthetic numbers indicate respective LETs). The gamma-irradiation
was at 60Co-gamma-source at KURRI, and those heavy
ion beams were obtained from HIMAC at NIRS. The
irradiated cells were embedded into agarose plugs, andFig. 1. The yields of clustered DNA damage (CDD)
in the irradated cells.

	 then the plugs were treated with endonuclease III and Fpg
for CDD containing oxidative pyrimidine and purine leisions, respectively. Finally, the plugs were subjected tostatic field agarose gel electrophoresis (SFGE) to estimate CDD yields.
Also, we analyzed isolated DNA damage including
oxidative base lesions by aldehyde reactive probe (ARP).
The cultured cells were irradiated by those radiations, andthe chromosomal DNA was extracted from the irradiatedcells by NaI. The DNA were treated by the appropriate
DNA glycosylases mentioned above, for identification ofbase lesions and then modified by ARP. Finally, we analyzed the ARP- modified DNA by ELISA-like procedure.
The radio-biological effectiveness of the irradiated cells
was estimated by colony forming assay. The detail ofanalysis procedures was on our previous report [1].
RESULTS: SFGE analyses showed that the total CDD
decreased in the elevation of the LETs (gamma > carbon> silicon > argon > iron) (Fig. 1). Similarly, the total isolated DNA damage decreased in elevation of the LETs
(Fig. 2). These results conform our previous result of
DNA solution [1]. On the other hand, colony forming
assay showed that the radio-biological effectiveness corresponded to the LET-elevation (data not shown). The
present result indicates that the relationship between
yields of DNA damage including CDD and radio-biological effectiveness is imbalance. Thus, CDD
seems to contribute the effect of heavy ion beams with its
some characters other than the quantity. For instance, wemust consider the importance of the quality, namely, thevariation of constituent lesions and the distribution in a
CDD for repair efficiency.
REFERENCES:[1] Terato H, et al., J Radiat Res 49 (2008) 133-146.

Fig. 2. The yields of isolated DNA damage in the irradated cells.

採択課題番号 24015	
  放射線によって生じる生体構成分子の損傷構造の特異性と生体影響機構の解析（佐賀大・総合分析セ）寺東宏明（京大・原子炉）齊藤	
  毅

共同通常

CO6-5 NAA for Trace Elements in Scalp Hair of Patients with ALS (the second report)
T. Kihira, I. Sakurai, S. Yoshida, I. Wakayama, K. Taka1
miya1, Y. Nakano1, R. Okumura1, Y. Iinuma , K. Iwai2,
3
4
4,5
K. Okamoto , Y. Kokubo and S. Kuzuhara
Department of Health Sciences, Kansai University of
Health Sciences (KUHS)
1
Research Reactor Institute, Kyoto University
2
Faculty of Nursing, KUHS
3
Department of Public Health, Aichi Prefectural College
of Nursing and Health4
Department of Neurology, Mie University Graduate
School of Medicine
5
Department of Medical Welfare, Suzuka University of
Medical Science

INTRODUCTION: A high incidence of amyotrophic
lateral sclerosis (ALS) has continued in the Koza /Kozagawa / Kushimoto (K) area in the Kii Peninsula ofJapan. We previously reported an elevation of transitional
metals in the scalp hair by NAA at KUR, and an elevation of urinary 8-hydroxydeoxyguanosine (8-OHdG), an
oxidative stress marker, of patients with ALS in this area
(K-ALS) [1]. Environment and lifestyle might play a rolein increasing oxidative stress on patients with K-ALS. To
examine the hypothesis that chronic Ca deficiency induce
an increase of absorption of toxic metals, and consequently increase metal-induced oxidative stress on neurons, we compared metal contents in the scalp hairamong patients with ALS in the K area, patients withsporadic ALS and residents in the Muro district.
EXPERIMENTS: Approximately 200 mg of hair was
obtained from patients with K-ALS, patients with sporadic ALS, residents in the Muro district and controls.
The hair samples were washed with 50 ml acetone, 50 ml
d.d.w. (3 times) and 50 ml acetone in sequence. The
samples were then dried in air. Approximately 30 mg of
the sample was weighed and double-wrapped in polyethylene films and subjected to NAA. The samples in polyethylene capsules were irradiated in Pn-1 of KUR (1000kW) for 2 minutes as short irradiations and for 120
minutes as long irradiations. As comparative standards,
orchard leaves (NBS), a human hair standard (NIES,CRM No.13) and elemental standards were used. The
γ-ray spectroscopic measurements with a Ge detector
were performed repeatedly according to the protocol previously reported [2].

Fig 1. The V contents in hair samples were shown (ppb,
boxes indicate median values, 25 and 75 percentiles). 1:
controls, 2: residents in the K area, 3: patients with K-ALS,
4: patients with sporadic ALS (*: p < 0.05).

RESULTS: Hair samples from seven patients with
K-ALS, 10 patients with sporadic ALS, 86 K-residents
and 19 controls were collected between 2010 and 2012
and the contents of Ca, Al, Cu, Mn and V were analyzed.
The samples were not treated with chemical procedures
including perm. The contents of V (148.3 ± 277.4 ppb,
mean ± S.D.) and Mn (0.75 ± 0.66 ppm) of patients with
K-ALS were higher than those of the controls (19.2 ±
11.1, 0.22 ± 0.35, respectively, p < 0.05, Fig. 1). Some of
the K-residents showed high V in the hair; however, themean content (28.6 ± 27.9 ppb) was not significantly different from the controls.
DISCUSSION AND CONCLUSIONS: The present
results indicate that the contents of V and Mn in the scalp
hair of patients with K-ALS were elevated. These transitional metals may play a role in increasing oxidative
stress on patients with K-ALS.
Part of this report is submitted to 24rd International Symposium
on ALS/MND held at Milan, Dec. 2013.

REFERENCES:[1] Kihira T, et al. Internal Medicine (2013), in press.
[2] Kihira T, et al. KUR Report (2012).

採択課題番号 24018 紀伊筋萎縮性側索硬化症における金属イオンと酸化的ストレス障害 共同通常
（関西医療大学･保健医療学部）紀平為子、吉田宗平、若山育郎、櫻井威織（京大・原子炉）高宮幸一、中野幸廣、奥村 良

CO6-6

Mechanism Involved in Tumor Tissue of Colon 26 Carcinoma-Bearing Mice
Irradiated with Neutron in the Presence of BSH-Appended Polyamine

T. Nagasaki, R. Kawasaki, M. Sakuramoto, H. Azuma, H.
Yanagie1, Y. Hattori2, M. Kirihata2, K. Ono3, S. Masunaga3 and Y. Sakurai3
Graduate School of Engineering, Osaka City University
1
Graduate School of Engineering, The University of Tokyo
2
Research Center for BNCT, Osaka Prefecture University
3
Research Reactor Institute, Kyoto University

INTRODUCTION: Recently, we synthesized a novel
polymeric boron carrier based on biodegradable polyamine, -poly-L-lysine, followed by cross-linking and modified with the boron cluster, BSH (BPP, Fig. 1) [1]. Since
this polymeric 10B carrier has anionic zeta-potential, polyion complexes with cationic polymer (nEG-PLL, Fig.1)
afford nanoparticles suitable for safe and effective delivery into tumor tissues due to Enhanced Permeability andRetention (EPR) effect. The neutron-irradiation experiment was carried out with the complex in colon 26 carcinoma-bearing mice. Significant depression effect for
the tumor proliferation was observed. Herein, mechanismof tumor growth suppression was assessed using
well-characterized TUNEL assay.
EXPERIMENTS: Colon 26 cells (8 x 105 cells) weretransplanted into a left thigh of mice (4 weeks old
BALB/c, male). After 10 days of transplantation, BPP
complex (BPP/23EG-PLL/9EG-PLL=8/4/1 w/w/w) solution was injected via tail vein at a dose of 4.0 mg 10B/kg(400 ppm of 10B concentration; 200 L). Twelve hours
after injection, neutron irradiation (4 x 1012 fluence/cm2)
was carried out at Kyoto University Reactor (1 MW, 90min). The mice were sacrified 24 h and 48 h after neutron

Fig. 2. TUNEL staining of tumors irradiated with neutron in
the absence (A) and presence (B) of administrated BPP complex.

irradiation, and the tumor tissues were taken out andfixed by freezing with compound. Ten m sections oftumor were stained with terminal deoxynucleotidyltransferase-mediated ‘nick-end’ labeling (TUNEL) using
a In Situ Cell Death Detection Kit, Fluorescein (Roche).
RESULTS AND DISCUSSION: When tumor-bearing
mice injected with BPP complex was irradiated with neutrons, significant depression effect for the tumor proliferation was observed (date not shown). Moreover, TUNEL
staining of thin sections of tumor tissues after injection of
BPP complex and neutron irradiation showed the DNA
fragmentation (Fig. 2). After 48 h of irradiation highest
DNA fragmentation was observed. These results indicated that BNCT with BSH-appended polyamine induced
apoptosis of tumor cells in vivo.

REFERENCE:[1] M. Umano et al., Appl. Radiat. Isot., 69 (2011)1765-1767.

Fig. 1. BSH-appended polyamine and based polyamines.

採択課題番号 24022

ホウ素クラスター修飾ポリアミンの腫瘍集積性および中性子捕捉反応効率評価

共同通常（阪市大院・工）長﨑 健、東 秀紀、李 家暐、鹿子嶋祐太、林高一郎、河崎 陸、櫻本昌士、
湯川寛子（東大院・工）柳衛宏宣（阪府大・BNCT 研）切畑光統、服部能英、門野尚之（京大・原子炉）小野公二、増永慎一郎、櫻井良憲

CO6-7

A New Reagent for Disulfide-Coupled Protein Folding

M. Okumura1,4, Y. Hidaka2, G. Inoue1, N. Fujii3, N.Fujii3,and H. Yamaguchi1
1School of Science and Technology, Kwansei Gakuin
University
2
Graduate School of Science and Engineering, Kinki
University
3
Research Reactor Institute, Kyoto University
4
Institute of Multidisciplinary Research for Advanced
Materials, Tohoku University

INTRODUCTION: The disulfide bond(s) play a critical role in the stabilities of their tertiary structures for the
expression of biological activities. The disulfide bondformation thermodynamically and kinetically controls
under redox conditions [1], for instance, concerning therole of glutathione, cystein, protein disulfide isomerase(PDI), and PDI oxidase. These catalyze the formation,
breakage, and isomerization of disulfide bond(s) in vivo
or in vitro. Recently, we demonstrated that a positively
charged redox reagent is preferred for accelerating disulfide-exchange reactions, as evidenced by the fact that thefolding recovery is greater than that for a typical redox
system [2,3,4]. Although the disulfide bond formationand the tertiary structure of a target protein are affectedby redox molecules, the nature of the redox environment
related to protein folding remains a matter of debate. The
objective of this research is to elucidate the disulfide-coupled folding mechanism by de novo designed
peptides as a redox molecule.
EXPERIMENTS:
Peptide Synthesis- The peptides were synthesized by
the Fmoc solid-phase method using a PSSM-8 peptide
synthesizer (Simadzu, Kyoto). The resulting peptides,
containing several cystein residues, were air-oxidized toform an intramolecular disulfide bond and the productwas purified by RP-HPLC (Hitachi High-Technologies
Corporation, Tokyo). The purified peptides were confirmed by MALDI-TOF/MS (Bruker Daltonics, Kanagawa) and stored in 0.1 M Tris/HCl buffer (pH 8.0) at room
temperature until used.
Preparation of reduced/denatured proteins- The reduced/denatured proteins were prepared according topreviously method [2], that is, purchased materials dissolved in 0.1 M Tris/HCl (pH 8.3) containing 20 mM
dithiothreitol and 8 M urea, and the supernatant was allowed to stand for 3 h for room temperature. The mixture
was then dialyzed against 10 mM HCl and lyophilized.
Micrococcus luteus assay- The refolding reaction was
performed in 0.1 M Tris/HCl pH 8.0 buffer containing
1.0 mM GSH and 0.2 mM GSSG in the presence or absence of 1 mM synthesized peptide (peptide A or B). A
Micrococcus luteus suspension (0.5 mg/mL) in 50 mM

phosphate buffer pH 6.5 was prepared to determine thebacteriolysis activity. The bacteriolysis reaction wasstarted by mixing 10 L the refolded lysozyme solution
and 1 mL a Micrococcus luteus suspension, and wasquenched by adding a quenching solution containing 0.5
M iodoacetic acid, 1 M KOH, 1 M Tris/HCl buffer pH
7.0 [3,4]. The light scattering intensities of the reaction
mixtures were measured at 600 nm.
Oxidative folding analyses- The denatured/reduced
proteins were dissolved in 0.1 M Tris/HCl pH 8.0 in thepresence of 2 mM reductant and 1 mM oxidant at room
temperature for 48 hr, as described previously [2,3]. All
solutions used in the folding experiments were flushedwith N2 gas. The reaction mixtures were removed at several time points, quenched with an equivalent volume of
1 M HCl [2,3], and separated by RP-HPLC. The HPLC
fractions were analyzed by MALDI-TOF/MS.
RESULTS: To estimate the efficiency for coupledprotein folding using the designed redox reagents, weemployed to lysozyme, bovine pancreatic trypsin inhibitor (BPTI), and prouroguanylin as a model protein. In thecase of lysozyme folding, peptide A or B accelerates thedisulfide coupled protein folding as compared to a typicalglutathione redox system, resulted in the increase of refolding yield.
By using HPLC analysis of disulfide-coupled folding of
BPTI we found that synthesized peptide A or B accelerates the formation and the isomerization of intra-molecular disulfide bonds: the folding intermediatesof BPTI were rapid rearrange to native form with correct
disulfide bonds pairings as compared to a typical glutathione redox system.
In general, several disulfide-containing proteins such as
lysozyme, BPTI, and RNaseA require redox condition to
fold correctly in vivo or in vitro, because proteins inwhich cysteine residues are involved in folding are folded
into the native conformation via the formation of
agent-mixed disulfide intermediates under redox conditions. It is therefore a complicated problem to choice thefolding condition with redox environment. This work
demonstrated a redox molecule is preferred in accelerating the disulfide coupled folding and the new approach iseffective in catalyzing the disulfide coupled protein folding. To elucidate the disulfide coupled protein folding
mechanism further, folding experiments in detail using
folding intermediates of prouroguanylin and BPTI are
already in progress.
REFERENCES:[1] M. Okumura et al., FEBS J., 279 (2012) 2283-2295.
[2] M. Okumura et al., FEBS J., 278 (2011) 1137-1114.
[3] M. Okumura et al., FEBS Lett, 586 (2012) 3926-3930.
[4] L. Ito et al., Protein J, 31 (2012) 499-530.

採択課題番号 24033
新規リフォールディング試薬の開発(関学大・理工)奥村正樹、井上 岳、山口 宏 (近大・理工)日高雄二(京大・原子炉)藤井紀子、藤井智彦

共同通常CO6-8Study of Effect on Elemental Levels in Marine Samples Caused by
The Great East Japan Earthquake in Ishinomaki Area

M. Fukushima, Y. Nakano1, R. Okumura1 andA. Yoshihara
Department of Basic Sciences, Ishinomaki Senshu
University
1
Research Reactor Institute, Kyoto University

INTRODUCTION: In 2011 March 11, great earthquake and Tsunami attacked Pacific side of Main Island,
Japan. Tsunami brought great amount of marine sediment to land, and after Tsunami we found the geographical feature of ocean floor has changed. In this study,
we have analyzed multi-elements in marine samples including marine sediments, shellfishes, marine invertebrates by neutron activation analysis to compare themwith elemental levels of samples obtained before The
Great East Japan Earthquake.
EXPERIMENTS: <Samples> Marine sediment, oysters, mussels, and sea squirt were collected in two different points (Higashimatsushima and Makinohama in
Oshika Pensula) in Sendai Bay. Marine sediment wasdried for 12 hours at 60 ℃. Soft tissues of oysters wereseparated from shells, hepatopancreas, muscles, gills, andmantles were separated each other, and freeze dried.
Dried samples were pulverized by mill. Soft tissues ofmussels were separated from shells, freeze dried, andpulverized by mill. Sea squirt was washed with tap water,
freeze dried, and pulverized by mill. <Neutron Activation Analysis> 0.3-0.5 g of dried samples were heatsealed doubly by cleaned polyethylene seat, and irradiated with several kinds of Standard Reference Materials(SRM). Irradiation was done for 12 seconds by Tc-Pn
or Pn, 1 minute by Tc-Pn, 20 minutes by Pn, and 4 hours
by Pn. After appropriate cooling time, gamma rays ofirradiated samples were measured by Ge detector. For
12 second irradiation, Compton suppression system(CSS) was used for measuring. Elemental levels were
calculated from the specific sensitivity of SRM.

Fig. 1 Gamma spectrum of sea squirt by CSS after 12 second irradiation.

採択課題番号 24068

RESULTS: Typical gamma spectrum by CSS is shownin Fig. 1, and gamma spectrum by normal Ge detector isshown in Fig. 2. By using CSS, it is obvious the background level for lower energy range has lowered. Elemental levels are shown in Table 1 for several samples
for the example without error. We have analyzed several elements in hepatopancreas of oysters from same collection point before The Great East Japan Earthquake.
The levels were 0.15, 198, 0.13, 0.03, and 477 ppm (dryweight base) for Co, Fe, Rb, Sc, and Zn, respectively.
By comparing them each other, we can say that Fe levelchanged lower and Rb level changed higher, and otherelemental levels did not change. We will continue theanalysis of these samples for further work.

Table 1. Elemental levels of samples obtained by NAA(μg/g, dry weight).
Element

marinesediment

Sea squirt(Ciona savignyi)

oyster, hepatopancreas

Al

19000

12300

ND

Br

64

226

188

Co

8.55

2.56

0.23

Cs

2.6

0.76

0.03

Fe

31700

6720

94.4

Mn

237

157

ND

Rb

45.1

12.2

3.8

Sc

5.5

32

0.02

Zn

93.7

77.2

488

Acknowledgement:
We thank to Mr. Shigeru Watanabe and his family, Dr.
Takeshige Matsutani in Ishinomaki Senshu University for
their help for collecting samples.

Fig. 2 Gamma spectrum of sea squirt by normal Ge detector systemafter 12 second irradiation.

石巻地域における東日本大震災による元素レベルの影響に関する検討（石専大・理工）福島 美智子、吉原 章（京大・原子炉）中野 幸廣、奥村 良

共同通常Evaluation of Time-Dependence of the Concentration of Gadolinium-BasedContrast Agent, Manganese, and Zinc in Normal and Nephrectomized Mice

Concentration (ppm)

70
60
50

40
30
20

70
60
50

40
30
20

10

10

0

0

Normal

1day

Model

7day

Normal

Model

3day

Normal

Model

1day

Normal

採択課題番号 24080

80

NormalRESULTS AND DISCUSSION: Almost of all the selected tissues in nephrectomized (model) mice showed
high Gd concentration compared to normal mice during
the experimental period. Gd concentration decreased withtime except for the femur in model mice, in which Gd
increased for 7 days (Fig.1). Due to the bone seeking
property of Gd and the low rate of the renal metabolism
compared to normal mice, Gd seems to accumulate in the
femur in the model mice at a higher concentration.

Gd-Femur
90

80

Model

EXPERIMENTS: Forty-one male 8-week-old ICR
strain mice were used in this study. Twenty-four mice
were housed as normal, while another seventeen mice
were partially nephrectomized as pseudo renal impairment model [6]. Half of each group was administered 2.5mmol Gd/kg b.w. Omniscan (gadodiamide) intravenously.
Mice were sacrificed and dissected 1, 3, 7 days after administration according to the experimental conditions.
Blood samples were collected, and the femur, kidney, andliver were excised and weighed. The samples were
freeze-dried and sealed into polyethylene bags for NAA.
The sealed samples and standard of gadolinium were
irradiated in Pn-3 for 30 s and in Pn-2 for 1 h, for shortand long half-life radioisotopes production, respectively.
The distribution of Gd and concentration of minerals
were determined by -ray spectrometry.

Gd-Skin90

NormalINTRODUCTION: Gadolinium (Gd)-based contrast
agents (GBCAs) have been used in medical magnetic
resonance (MR) imaging. In particular, human nephrogenic systemic fibrosis (NSF) has been reported in patients with severe renal insufficiency treated with
Gd-based contrast agent [1]. A depletion of endogenous
Zn ion is proposed as one of the reasons for development
of NSF [2, 3]. On the other hand, there also reported nocorrelation between Zn and NSF [4]. Our recent findings
also showed no correlation of Zn with both GBCA andnephrectomized situations [5]. However, those results
were obtained from only one time point after GBCA administration (2 days) and, therefore, the relation between
Gd and trace elements in vivo is still unclear.
In this study, we evaluated the time dependence of both
the distribution of Gd and concentration of some minerals
by neutron activation analysis (NAA) in selected tissuesof four mice under different experimental conditions.

3day

7day

Fig. 1. Time dependence of the concentration of Gd in theskin and femur of normal and nephrectomized mice after
GBCA administration. White and gray bars represent
normal mice and nephrectomized mice, respectively.
Zn-Skin

Mn-Liver
40

2.5

35
2

Concentration (ppm)

School of Health Sciences, College of Medical, Pharmaceutical and Health Sciences, Kanazawa University
1
Research Reactor Institute, Kyoto University

The concentration of Mn in the liver and Zn in the skinare shown in Fig. 2. The liver of Gd-administered model
mice showed increased Mn concentrations compared tonormal mice. Zn concentration in model mice was highcompared to other groups after 1 day of Gd administration. On the other hand, Gd-administered model mice
showed high Zn concentration after 7 days.
We evaluated the time-dependence of the concentrationof “exogenous” Gd and “endogenous” Mn and Zn. However, these results do not sufficiently provide statisticallywarranted representations of the biodistribution of “exogenous” Gd and the concentration of “endogenous” and“exogenous” trace elements in normal and model mice toclarify the correlation between Gd and trace elements.
Concentration (ppm)

K. Washiyama, T. Matsumoto, E. Shinohara, Y. Nakanishi, Y. Murasaki, R. Amano and K. Takamiya1

Concentration (ppm)

CO6-9

1.5

1

30
25
20
15
10

0.5

5
0

0

a

b

c
1day

d

a

b
3day

a

b

c

7day

d

a

b

c

1day

d

a

b

3day

a

b

c

d

7day

Fig. 2. Time dependence of the concentration of Mn inthe liver and Zn in the skin of normal and nephrectomized mice after GBCA administration. White, black,and striped bars represent normal mice, model mice, andgroups of GBCA administration, respectively.
REFERENCES:[1] J. C. Weinreb and A. K. Abu-Alfa, J. Magn. Reson.
Imaging, 30 (2009) 1236-1239.
[2] E. S. Harpur et al., Invest. Radiol., 28 (Suppl 1)(1993) S28-S43.
[3] J. H. Wible et al., Invest. Radiol., 36 (2001) 401-412.
[4] H. Pietsch et al., J. Magn. Reson. Imaging, 30 (2009)374-383.
[5] K. Washiyama et al., KURRI Progress Report 2011,(2012) PR1-6.
[6] H. Pietsch et al., Invest. Radiol., 44 (2009) 226-233.

MRI 用ガドリニウム造影剤による副作用「腎性全身性線維症」の発症機序の解明に関する研究-アクチバブルトレーサー法の応用-（原子炉中性子を用いた微量元素分析）（金沢大・保健）鷲山幸信、松本高史、篠原絵里香、中西勇介、村崎祐一、天野良平（京大・原子炉）高宮幸一

共同通常CO6-10

Mössbauer Studies on Reversible O-O Bond Scission of Peroxodiiron(III) to
High-Spin Oxodiiron (IV)

M. Kodera, Y. Kawahara and S. Kitao1
Department of Molecular Chemistry and Biochemistry,
Doshisha University
1
Research Reactor Institute, Kyoto University
INTRODUCTION: Soluble methane monooxygenases(sMMO) are non-heme diiron enzymes catalyzing conversion of CH4 to CH3OH, one of the most difficult chemical
oxidation, via a dioxygen activation, where intermediate P,
peroxodiiron(III) generated by O2-binding to diiron(II), isconverted to active species Q, oxodiiron(IV) responsible for
the CH4 oxidation[1-3]. The diiron(IV) of Q is in a high-spin
S = 2 state.3 A di--oxodiiron(IV) diamond core structure is
proposed for Q from the short Fe•••Fe distance (2.5 Å),[4]but not established. The conversion of P to Q, however, has
not been clarified due to their instability [3,5] Thus, a functional model attaining conversion of peroxodiiron(III) to
high spin oxodiiron(IV) is useful for clarification of the
P-to-Q conversion mechanism.
Recently, we succeeded in spectral observation of the reversible O-O bond scission of peroxodiiron(III) to oxodiiorn(IV) with a bis-tpa dinucleating ligand 6-hpa (1,2-bis[2[bis(2-pyridylmethyl)aminomethyl]-6-pyrid-yl]ethane)[6].
We report herein the Mössbauer measurements of the conversion of the peroxodiiron(III) to the oxodiiron(IV).
EXPERIMENTS: Preparation of a mixture of peroxodiiron(III) and oxodiiron(IV) complexes 2. To a solution of [Fe2(-O)(OH2)2(6-hpa)](ClO4)4 (1) (45.1 mg 36.3μmol) in MeCN (2.5 mL) was added 2.0 equiv of Et3N (29mM) at -40°C, and stirred for 5 min. To the solution wasadded 1.2 equiv of H2O2 (17 mM), and stirred for 2 min. The
solution turned dark green. To the resultant solution wasadded 20 mL of Et2O at -40°C, green solid precipitated. The
supernatant was decanted off, and the precipitate waswashed with Et2O several times at -40 °C. The green solid
was dried in vacuo. The isolated solid is stable at low temperature, and not changed several days at room temperature
under dark in the absence of any organic compounds potentially acting as reductant. Yield 31.3 mg (33.8 μmol, 93.1 %).
Anal. Calcd for C38H46N8Cl2O15Fe2: C, 43.99; H, 4.47; N,
10.80%. Found: C, 44.21; H, 4.32; N, 10.69%. The isolated
solid was used for various spectral measurements including
the Mössbauer spectra.
RESULTS: In the previous studies, the Mössbauer
spectra of the isolated solid 2 were recorded between 25 and295 K at zero field. The spectrum at 25 K is composed of
two quadrupole doublets with  = 0.351(3) mm/s, ΔEQ =1.635(5) mm/s and  = 0.132(9), ΔEQ = 0.438(2), as shownby the deconvolution spectra, the red and blue lines in Figure 1(a), respectively. The  and ΔEQ values of the red line
are close to 0.54 and 1.68 mm/s of -oxo--peroxodiiron(III) 5, 0.66 and 1.40 mm/s of -peroxo-di--benzoatodiiron(III) with sterically hindered trispyrazolylborate
HB(pz’)3,
and
0.53
and
1.68
mm/s
of
-peroxo-di--acetatodiiron(III) of hexpy ligand. Thus, thered line may result from a diiron(III) symmetrically bridged

採択課題番号 24092

by the peroxide. The  and ΔEQ values of the blue line areclose to  = 0.14-0.21 and ΔEQ = 0.53-0.68 mm/s of highspin S = 2 diiron(IV) in the active species Q of sMMO. The
high spin S = 2 iron(IV) complexes reported so far show the and ΔEQ values around 0.1 and 0.5 mm/s, respectively, but
low spin S = 1 iron(IV) relatively higher ΔEQ values 1-2
mm/s. Thus, the blue line must be due to a high spin S = 2
diiron(IV) with a symmetric structure. These results and allother spectral and analytical data shown above demonstrated
that 2 is composed of the -oxo--peroxodiiron(III) complex 2a and the high spin S = 2 -oxodioxodiiron(IV) complex 2b. Here, 2b is the first synthetic example of ahigh-spin S = 2 oxodiiron(IV).
raising the temperatures

recooling the same sample(a) 25K(e) 25K(b) 100K(f) 100K(c) 170K(d) 294K

Fig 1. Zero field Mössbauer spectra of 2, recorded by raising
the temperature at (a) 25, (b) 100, (c) 170, and (d) 294 K, andby recooling the same sample at (e) 25 and (f) 100 K. The blackline is the least-square fitting to the raw data, and the red andblue lines are the deconvolution spectra corresponding to-oxo--peroxodiiron(III) (2a) and -oxo-dioxodiiron(IV) (2b)complexes.

In this study, further, we tried to measure the Mössbauer
spectra under applied magnetic field conditions. However, it
was not successful because upon preparation of the KBr diskof the measurement sample, the peroxodiiron(III) complex
was decomposed to a structurally unknown diiron(III) complex. So, we may need to improve the sampling method. In
this year, we will try to optimize the sampling method andcarry out some more experiments of the Mössbauer measurements under the applied magnetic field.

REFERENCES:[1] Tinberg, C. E.; Lippard, S. J. Acc. Chem. Res. 2011,
44, 280-288.
[2] Friedle, S.; Reisner, E.; Lippard, S. J. Chem. Soc. Rev.
2010, 39, 2768-2779.
[3] Wallar, B. J.; Lipscomb, J. D. Chem. Rev. 1996, 96,
2625-2658.
[4] Shu, L.; Nesheim, J. C.; Kauffmann, K.; Münck, E.;
Lipscomb, J. D.; Que, L. Science 1997, 275, 515-518.
[5] Liu, K. E.; Valentine, A. M.; Wang, D.; Huynh, B. H.;
Edmondson, D. E.; Salifoglou, A.; Lippard, S. J. J. Am.
Chem. Soc. 1995, 117, 10174-10185.
[6] M. Kodera, Y. Kawahara, Y. Hitomi, T. Nomura, T.
Ogura, Y. Kobayashi, J. Am. Chem. Soc., 2012, 134,
13236.

パーオキソ二核鉄（Ⅲ）とトリオキソ二核鉄（Ⅳ）の相互変換に関するメスバウアースペクトルによる研究（同志社大院理）小寺政人、河原由佳（京大・原子炉）瀬戸誠、北尾真司

共同通常Determination of Trace Elements in Organs and Tissues of Zn-Deficient MiceM. Yanaga, A. Oishi, K. Sera1, R. Okumura2 and Y. Iinuma2
Graduate School of Science, Shizuoka University
1
Cyclotron Research Center, Iwate Medical University
2
Research Reactor Institute, Kyoto University

INTRODUCTION: Previously, we reported that the
Co content increased significantly in all the organs andtissues of Zn-deficient mice, which had been fed with
Zn-deficient diet for 1 week, 3 weeks, or further period from 3 or 8-week old, compared with those of mice
fed with control diet, and that this fact suggested thepartial substitution of Co with Zn in their metal proteins or other metal-bound compounds.[1-3] In thepresent work, concentrations of trace elements in organs and tissues of mice were determined in order toexamine whether cobalt functions as a substitute for
zinc.
EXPERIMENTS:

Animals and preparation of samples Male mice of
the ICR/jcl strain, 8-week old, were divided into twogroups.
One group was fed with Co-enriched
Zn-deficient diet (the diet was made with raw materials for Zn-deficient diet and Co as CoCl2) and ultra
pure water (Co-enriched Zn-def. mice) for one or three
weeks.
The other one group was fed with
Cu-enriched Zn-deficient diet (the diet was made with
Cu as CuSO4·5H2O) and the same water (Cu-enriched
Zn-def. mice).
After the treatment, about 1 mL of blood wascollected from heart under diethyl ether anesthesia,
and then, 5 organs and tissues such as liver, kidney,pancreas, testis, and bone were removed. The blood
samples were centrifuged and the serum was harvested
for PIXE analysis. The removed organs and tissues
were weighed, freeze-dried, weighed again andgrounded. Each sample was doubly wrapped in polyethylene film for INAA (Instrumental Neutron Activation Analysis).
PIXE Analysis One hundred L of the serum was
dropped on a backing film (polypropylene) and directly irradiated with proton beam at Nishina Memorial Cyclotron Center, Japan Radioisotope Association.
The spectra were analyzed with the standard-freemethod. [4, 5]
INAA The samples in polyethylene capsules were
irradiated in Pn-1 for 90 seconds and for 2 hours, forshort and long irradiation, respectively. The -ray
spectroscopic measurements with an HPGe detector
were performed repeatedly for the short-irradiatedsamples: the first measurements for 120 seconds after
decay time of 5 - 10 minutes and the second one for
250 seconds after 80 - 120 minutes. The long-irradiated samples were measured for 3 - 24 hours
after an adequate cooling time (60 - 80 days).
RESULTS: As results of PIXE analysis of the serum
samples, Co concentration of Co-enriched Zn-def. mice
was much risen whereas Cu concentration of

Cu-enriched Zn-def. mice was not higher than that of
Co-enriched Zn-def. mice. Similar trend was foundfor all the organs and tissues investigated by INAA.
On the other hand, Zn concentration in pancreases ofmice which were fed with Co-enriched Zn-deficient
diet for three weeks was slightly restored as shown in
Fig. 1. This might indicate that some kinds ofmechanisms which enhance the intake of Co in all theorgans and tissues of Zn-def. mice could be operated,
and that Co would function to disturb that Zn flowedout.
50

Zn concentration / (g/g)

CO6-11

Control
Zn-def.
Co enriched Zn-def.
Cu enriched Zn-def.

40
30
20
10
0

0

1 week treatment

3 weeks treatment

4

Fig. 1 Zn concentration in pancreases of mice
REFERENCES:[1] M. Yanaga, M. Iwama, K. Takiguchi, M. Noguchi
and T. Omori, J. Radioanal. Nucl. Chem., 231 (1998)
187-189.
[2] M. Yanaga, M. Iwama, K. Shinotsuka, K.
Takiguchi, M. Noguchi and T. Omori,J. Radioanal. Nucl. Chem., 243 (2000) 661-667.
[3] M. Yanaga, H. Wakasa, T. Yoshida, M. Iwama,
K. Shinotsuka, M. Noguchi and T. Omori,J. Radioanal. Nucl. Chem., 245 (2000) 255-259.
[4] K. Sera, S. Futatsugawa, K. Matsuda and Y. Miura,
Int’l Journal of PIXE, 6(1996) 467-481.
[5] K. Sera, S. Futatsugawa, S. Hatakeyama, Y. Saitoh
and K. Matsuda, Int’l Journal of PIXE, 7(1996)
157-170.

採択課題番号 24101
亜鉛代用金属としてのコバルトの投与（静岡大院・理）矢永誠人、大石歩実 （岩手医大・サイクロ）世良耕一郎（京大・原子炉）奥村 良、飯沼勇人

共同通常