CO6-1

Neutron Activation Analysis for Trace Elements in Scalp Hair of Patients with ALS

T. Kihira, I. Sakurai, S. Yoshida, I. Wakayama, K.
Takamiya1, Y. Nakano1, R. Okumura1, S. Morinaga2,
S. Wada2, K. Iwai2, K. Okamoto3, Y. Kokubo4 and S.
Kuzuhara4,5
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 amyo-

trophic lateral sclerosis (ALS) and parkinsonism-dementia complex (PDC) has been found in
the Koza/Kozagawa/Kushimoto (K) area andHohara area of the Muro district in the Kii
Peninsula of Japan. Environmental research
revealed that drinking water in the K area
showed low contents of Ca and Mg and the serum of residents and patients with ALS in the
K area also showed low contents of Ca [1]. It
is reported that Ca deficiency induces distributional changes of other metals in bones and soft
tissues by secondary hyperparathyroidism. We
speculated that a chronic Ca deficiency might
induce an increase of absorption of toxic metals,
and consequently increase metal-induced oxidative stress on neurons [2] and play a role inthe development of ALS and PDC in the Muro
district. To examine this hypothesis, we investigated the metal contents in the scalp hair ofpatients with ALS or PDC and residents in the
Muro district.
EXPERIMENTS:
Approximately 200 mg ofhair was obtained from respective patientswith ALS or PDC in the Muro district, 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 wasweighed and double-wrapped in polyethylene

採択課題番号 23007

films and subjected to NAA. The samples inpolyethylene capsules were irradiated in Pn-1
of KUR (1000 kW) for 2 minutes as short irradiations and for 120 minutes as long irradiations. As comparative standards, orchard
leaves (NBS) as well as elemental standards
for Ca, Mg, Cu, Al, Mn, V, Br, Cl, S, Zn, and Hg
were used. The -ray spectroscopic measurements with a Ge detector were performed repeatedly. The measurement time was 200 seconds for the short-lived nuclides after a cooling time of 110-320 seconds, and for the
long-lived nuclides, it was 5000-6000 seconds
after cooling for approximate one month.
RESULTS: 68 residents in the Muro district, 6
patients with ALS and 5 patients with PDC
from the Muro district, and 22 residents fromcontrol area participated in this research. The
contents of Ca, Mg, Al, Cu, Mn, Cl, Hg, V, S and
Zn were determined in the hair samples. The
Ca content was significantly influenced by hairtreatments. The Mg content in hair samples
from patients with ALS or PDC in the Muro
district was significantly higher than those ofcontrols (Fig.1).
ppm

Case categorycaFig. 1. The Mg content in hair samples was shown (ppm,
Mean +- SE). 10: controls without hair treatments, 11: controls with hair treatments, 20: residents in the K area without hair treatments, 21: residents in the K area with hairtreatments, 40: patients with PDC without hair treatments,
50: patients with ALS without hair treatments.

Part of this report is submitted to 23rd International Symposium on ALS/MND held at Chicago, Dec. 2012.
REFERENCES:[1] Kihira T, et al. NEUROLOGICAL MEDICINE
2010; 73: 507-51[2] Rothstein JD. Ann Neurol 2009; 65 Suppl 1:
S3-9

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、奥村良共同通常

&2

Structure6pecificity Rf 5adio-Induced%iomolecule'amageDnd,ts(ffectRadio-Biological Consequence

H. Terato and T. Saito1
Analytical Research Center for Experimental Sciences,
Saga University
1
Research Reactor Institute, Kyoto University
INTRODUCTION: Ionizing radiation causes various
deleterious effect for living organisms. The effect is derived from damaging of biological molecules with ionizing radiation. Among numerous biological molecules,
deoxyribonucleic acids (DNA) seem to be the most important ones as the only one genetic material. An ionizing
radiation passes through (or along) the target DNA
molecule as a beam. As passing, it leaves multiple damage close to the track. Such damage accumulation is termed as clustered DNA damage (CDD). CDD is considered as a damage aggregation containing two or morelesions in the two or three helices. The complex structureof CDD might lead to be its less repairability and highinhibition for DNA replication. CDD seems to be a major
factor responsible to severity of radiobiological consequence [1]. In this study, we estimated the yields of CDD
in the irradiated cultured cells to clarify the function of
CDD in radiobiological effect.
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 embedded in agarose plugs were analyzed
on agarose gel electrophoresis. For estimation of the total
CDD, we summed DNA-double strand breaks (DSB) andoxidative base lesion clusters (BLC). The detail of analysis procedure was based on our previous report [2].

RESULTS: The yields of total cellular CDD decreased
as LET of the radiations grew (Fig. 1). On agarose gelelectrophoresis, total damaged fractions of the chromosomal DNA in irradiated cells were 2.8%, 1.9%, 1.7%,
1.4% and 0.65% for gamma-ray, carbon, silicon, argon
and ion particle beams, respectively. Similarly, the yieldsof total isolated DNA damage shows parallel pattern for
the LETs (Fig. 2). Isolated damage were analyzed by
ARP [2]. The total isolated DNA damage in the irradiatedcells were 0.315, 0.206, 0.141 and 0.122 sites 10 bp-1
Gy-1 for gamma-ray, carbon, silicon and argon particle
beams, respectively. The data of isolated damage for ironparticle beam is now under analysis. On the other hand,the survival fractions of the irradiated cells reduced for
LET increasing. The D10 for gamma-ray, carbon, silicon,
argon and ion particle beams, were 4.8, 3.7, 2.6, 2.4 and
2.2 Gy, respectively, which were from colony forming
assay. Those results indicate that the yields of radiation
DNA damage even the clustered DNA damage are notdirectly bound up with the severity of radiobiologicaleffect. We previously demonstrated the similar resultswith in vitro analyses for plasmid DNA and phage DNA
irradiated with various ionizing radiations including
heavy ion beams [2]. Many other studies also indicatedsimilar correlation between yields of DNA damage andLET of ionizing radiation, too. Therefore, we need toconsider another factor for the nature of clustered DNA
damage other than the quantity. These are thought to be
the structure, repair efficiency and others.
Conclusively, the yields of radiation CDD showed
inversely proportional to their LETs in irradiated cells. It
suggests that yields of CDD are not simply responsible toseverity of radiobiological consequence.
REFERENCES:[1] H. Terato and H. Ide, Biol. Sci. Space, 18 (2004)
206-215.
[2] H. Terato et al., J. Radiat. Res., 49 (2008) 133-146.

	
 

0.4
Total Isolated Damage(10 bp-6 Gy-1)

Total CDD (% Gy-1)

3.0
2.5
2.0
1.5
1.0
0.5
0.0
Gamma Carbon

Silicon

Argon

Iron

0.3
0.3
0.2
0.2
0.1
0.1
0.0
Gamma

Fig. 1. The yields of clustered DNA damage (CDD)
in the irradated cells.

Carbon

Silicon

Argon

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

採択課題番号 23011	
  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‫౒ޓޓޓ‬หㅢᏱ‫↢ޓޓޓޓޓޓޓޓޓޓ‬૕ᓇ㗀ᯏ᭴ߩ⸃析（佐賀大・総合分析セ）寺東宏明、（京大・原子炉）齊藤	  毅Determination of Trace Elements in Organs of Zn-deficient MiceM. Yanaga, H. Tanaka1 and Y. Nakano2
Radiochemistry Research Laboratory,
Faculty of Science, Shizuoka University
1
Department of Chemistry,
Graduate School of Science, Shizuoka University
2
Research Reactor Institute, Kyoto University

INTRODUCTION: Zinc is one of the most importantessential trace elements and it interacts with manyother trace elements in living organisms.
Ourprevious works have shown that the Co content
increased significantly in all the organs and tissues ofZn-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 ofmice fed with control diet, and that this fact suggested
the partial substitution of Co with Zn in their metal
proteins or other metal-bound compounds [1-4] . In
the present work, concentrations of trace elements inorgans of mice were determined in order to compare
the effect of the zinc dosage of several kinds ofchemical form on zinc deficiency.
EXPERIMENTS:
Animals and samples Male mice of the ICR/jcl strain,
8-week old, were divided into five groups. One
group was fed with Zn-deficient diet and ultra purewater (Zn-def. mice) for two weeks. The other one
group was fed with control diet (the diet was madewith raw materials for Zn-deficient diet and Zn as zinccarbonate, basic) and the same water for two weeks.
Remaining three groups were fed with Zn-deficient
diet for a week. Then, they were fed with control
diet described above or the other two kinds ofspecially prepared control diet in which Zn was added
as zinc gluconate or zinc sulfate.
After the treatment, their livers, pancreata, andtestes were removed under diethyl ether anesthesia.
The removed organs were homogenized with HEPES
buffer. In this process, the pancreata and testes ofevery four mice of each group were together
homogenized because each pancreas or testis is too
small for analysis.
Cytosolic fractions wereseparated by differential centrifugation at 105,000g
for 65 min.
They were weighed, freeze-dried,weighed again and grounded. Each sample (10 - 50mg) was doubly wrapped in a polyethylene film andsubjected to INAA(Instrumental Neutron Activation
Analysis).

INAA The samples in polyethylene capsules were
irradiated in Pn-1 for 6 minute and for 4 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 – 300 seconds
after decay time of 5 - 10 minutes and the second one
for 250 - 900 seconds after 60 - 150 minutes. The
long-irradiated samples were measured for 6 - 24hours after an adequate cooling time (15 - 80 days).
RESULTS: Content of ten elements, Na, Mg, Cl, Mn,
Fe, Co, Cu, Zn, Se and Rb, were determined. Zinccontent in cytosolic fraction of livers and testes ofZn-defcient mice were not lower than those of control
mice or the mice which were fed with Zn-added dietafter feeding with Zn-deficient diet. The content incytosolic fraction of pancreatic cells of Zn-deficient
mice were distinctly lower than those of the othermice. On the other hand, the data of Co content
indicates that one-week zinc dosage was insufficient
for recovery from zinc deficiency as shown in Fig. 1.
0.05

0.04

Co content / g

CO6-3

0.03

0.02

0.01

0.00

1

2

3

4

5

Fig. 1 Cobalt content in cytosolic fraction of livers of various kinds of mice;
1: Zn-def. diet
2: Zn-def.diet + control diet 3: Zn-def. diet + ZnSO4 included diet
4: Zn-def. diet + zinc gluconate
5: control diet

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] T. Ogi, Y. Kawamoto, H. Maetsu, M. Koike, T.
Ohyama, S. Enomoto, M. Noguchi, H. Suganuma
and M. Yanaga, Proc. Intern. Symp. on Bio-Trace
Elements 2002 (2000) 83.

採択課題番号 23023
亜鉛欠乏マウス臓器中の微量元素の分析（静岡大・理）矢永誠人 （静岡大院・理）田中宏宗 （京大・原子炉）中野幸廣

共同通常

CO6-4

New Reagents for Disulfide-Coupled Protein Folding

M. Okumura1,4, Y. Hidaka2, G. Inoue1, T. Maekawa1, L.
Ito1, 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
JSPS
INTRODUCTION: The disulfide bond formations areimportant not only for the folding of secreted proteinsand peptides, but also for the stabilities of their tertiary
structures for the expression of biological activities. Suchreactions are thermodynamically and kinetically related
to the redox reagents and their redox potential [1], forinstance, glutathione, cystein, protein disulfide isomerase(PDI), and their relatives (Dsb proteins). These reagentscatalyze both the formation and isomerization of disulfide
bonds in vivo or in vitro. Recently, we demonstrated that
positively charged redox reagents are preferred for accelerating the disulfide-exchange reactions, as evidenced by
the fact that the folding recoveries by using such reagentsare greater than that by using a typical redox system [2].
Although the formation of disulfide bonds and the tertiary structure of a target protein is affected by a designed
redox reagent, the nature of the redox environment its
relationship with protein folding remains a matter of debate. The objective of this study was to develop the effective disulfide-coupled folding method by using new
additives as a redox reagent.
EXPERIMENTS: Peptide Synthesis- Two kind of
peptides (peptide A and peptide B) were synthesized by
the Fmoc solid-phase method by using a PSSM-8 peptide
synthesizer (Simadzu, Kyoto). The resulting peptides,
containing two cystein residues, were air-oxidized toform an intramolecular disulfide bond and the productwas purified by RP-HPLC (Hitachi High-Technologies,
Tokyo). The purified peptides were dissolved in 0.1 M
Tris/HCl buffer (pH 8.0).
Preparation of reduced/denatured proteins- The reduced/denatured proteins were prepared according toprevious method [2], that is, proteins, hen egg whitelysozyme (HEWL), bovine pancreatic trypsin inhibitor(BPTI), and prouroguanylin, were dissolved in 0.1 M
Tris/HCl (pH 8.3) containing 20 mM dithiothreitol and
8 M urea, and the solutions were kept at room temperature for 3 hours. The reaction mixtures were then dialyzed against 10 mM HCl and lyophilized.
Kinetic analyses- The refolding reaction of HEWL
was performed in 0.1 M Tris-HCl (pH 8.0) buffer containing 1.0 mM GSH and 0.2 mM GSSG in the presenceor absence of 1 mM the synthesized peptide (peptide A or
B). Correctly folded HEWL has bacteriolysis activity, aMicrococcus luteus suspension (0.5 mg/mL) in 50 mM
phosphate buffer (pH 6.5) was prepared to measure theactivity. The bacteriolysis reaction was started by mixing
10 µL of the refolded HEWL solution and 1 mL of a Micrococcus luteus suspension, and was quenched at several time-points by adding a quenching solution containing
0.5 M mono-iodoacetate, 1 M KOH, 1 M Tris/HCl buffer(pH 7.0). 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) andallowed to undergo folding in the presence of 2 mM reductant and 1 mM oxidant at room temperature for 48
hours, as described previously [2]. All solutions used in
the refolding experiments were flushed with N2 gas, andthe reactions were carried out under an atmosphere of N2
in a sealed vial. The reaction mixtures were sampled atseveral time-points, quenched with an equivalent volumeof 1 M HCl [2], and separated by RP-HPLC. The HPLC
fractions were analyzed by MALDI-TOF/MS after lyophilization.
RESULTS: To estimate the effects of the synthesizedpeptides on protein folding, we employed HEWL, BPTI,
and prouroguanylin as model proteins. Refolding yields
of HEWL by using peptide A or B were greater than thatby using a typical glutathione redox system, suggestingthat folding velocity increased in the presence of thesynthesized peptides.
HPLC analyses of disulfide-coupled folding of BPTI
showed that the reduced/denatured and the scrambled-disulfide species (folding intermediates) of BPTI
rapidly converted to native form with correct disulfide
bonds as compared with glutathione redox system. It isconsidered that the synthesized peptides A and B accelerated the formation and the isomerization of intra-molecular disulfide bonds.
In general, an appropriate redox condition is necessary
for correct folding of disulfide-containing proteins thathave multiple disulfide bridges, because these proteins
fold into the native conformations via the formation ofcomplex disulfide intermediates. It is therefore a complicated problem to choose the suitable folding condition
with redox environment. In our study, we demonstrated
that the new redox molecules, peptide A and B, arepreferred for accelerating the folding reaction and that the
new method using them is effective in forming the native
conformation of proteins. To investigate the further folding mechanism by using the new reagents, analysis using
folding intermediates of prouroguanylin is in progress.
REFERENCES:[1] J.L. Arolas, et al., Trends Biochem Sci., 31 (2006)
292-301.
[2] M. Okumura et al., FEBS J., 278 (2011) 1137-1114.

採択課題番号 23049
新規リフォールディング試薬の開発
共同通常（関学大・理工、学振） 奥村正樹、 （近畿大・理工） 日高雄二、 （関学大・理工） 井上 岳、
前川拓摩、伊藤 廉、 （京大・原子炉） 藤井紀子、藤井紀彦、 （関学大・理工） 山口 宏

CO6-5 Combination Therapy of BNCT and PDT Sensitized by A Boron-Porphyrin Derivative in a
Glial Cancer Cell Line

Department of Tumor Pathology, Faculty of Medicine,
University of Fukui
1
Department of Neurosurgery, Graduate of Human Sciences, Tsukuba University
2
Research Reactor Institute, Kyoto University
3
A. N. Nesmeryanov Institute of Organoelement Compounds, Russia Federation
4
Blokhin National Cancer Center, Russia Federation
INTRODUCTION: Recently, the cancer treatment by
BNCT has been taken the clinical trial widely in Kansai
region and the near fields at the special institution, Research Reactor Institute of Kyoto University. Furthermore,
the compact generator of neutron beam will be progressed in future, especially against the malignant tumor
cases. It should stop it as the malignant tumor will repeat
the re-growth of it combined with other treatment for
example, photodynamic therapy (PDT) using the photosensitizer and a compact LD laser [1-2]. We had beendeveloped the pulsed two wavelength of 635 and 665
using 5-aminolevulinic acid (5-ALA) [3-4]. In this machine time (14th Dec. 2011 and 17th Jan. 2012), we experimented combination treatments of BNCT and PDT ofa glial cancer cell line (C6) using a boronated porphyrinderivative as the both sensitizer which was presented
from Russia Cancer Center and Institute ofOrganoelement Compounds.
EXPERIMENTS: The irradiated cell samples wereprepared with the 1.4M sensitizer (compound-B) solutions for 30 min incubation in a CO2 incubator before theboth irradiations (2.1Gy for 20 min of BNCT and
150J/cm2 for 17min of PDT, the duration time was 36 hr
between both irradiations). The dose of neutron beam
was finally calculated from the average value of the 7detectors of gold leaf and thermo-luminescence dosimeter on the 24 Eppendorf sample tubes. The behind andsides of sample were sealed with 5 enriched lithium-6
fluoride plats (2mm thickness). The percentage of thenecrotic cells stained with an apoptosis kit(annexinV-FITC and propidium iodide (PI), MBL Co.
Ltd., Nagoya) were analyzed by a flowcytometer (BDFACS-Canto-II type, BD Sciences, Co. Ltd., Tokyo) after
the 1 day incubation.
RESULTS and DISCUSSION: It was resulted that the
combination therapy of a tumor cultivated cell line (C6)cells were effectively damaged to induce the necrotic cell

採択課題番号23073

death because the damaged cells were stained at higher
percentages by propidium iodide as following graph in
the lower slide.

Group

Necrotic Cell Death Treated by BNCT and PDT
with Compounds-A and B
1

Necrotic Cell Death (%)

2

Different Treatment GroupN. Miyoshi, A. B. Bibin, T. Yamamoto1, A. Matsumura1,
H. Tanaka2, K. Ono2, V. N. Kalinin3, V. A. Ol'shevskaya3,
A. V. Zaitsev3 and A. A. Shtil4

3
4
6
7
8
9
11
12
13
14
0

10

20
30
40
Necrotic Cell Death (%)50(1) [Compound-A or -B]=1.4 M/ 2x105 C6 glial tumor
cells/ml for 30min and washed with PBS solution in the monolayer condition.
(2) The condition of the cells irradiated by Neutron was single
flowing cells in the medium solutions inside Eppen-dorf tube
(1.5ml).

1. Control (-Compound-B; -BNCT)
2. BNCT (+Compound-B))
3. Compound-B)
4. PDT (+Compound-B)
6. BNCT-PDT (+Compound-B)
7. PDT-BNCT (+Compound-B)
8. Compound-A
9. PDT (+Compound-A)
11. BNCT-PDT (+Compound-A)
12. PDT-BNCT (+Compound-A)
13. PDT (-Compound-A)
14. BNCT (-Compound-A)*Cell (C6: Glial Tumor of rat) Analysis
stained with PI-FITC by a flow
cytometer (BD FACSCanto-II) in
Fukui University.
**Laser irradiation Dose by 635, 665nm(28 J/cm2) in Fukui University.
***BNCT irradiation Dose: 0.2 Gy/[10B]
ppm by 1 MW. (Na 0.11, Cl 0.11) inKyoto University, which will be
estimatedmore
indetail([10B]=11.9ppm /2x105 cells) for 20 min23(Total Dose=2.0 Gy).

Especially, it was large effects only when the BNCT
treatment had done before the PDT one using the compound-B. The anti-cancer effect exchanged sequence wasno effect. It was estimated that the damaged cell analysis
was done after the incubation for over-night of the treatedcells which were recovering from the light (small) damages.
ACKNOLEDGEMENTS: I (N.M.) would like to thankthe JST foundation and the research reactor institute of
Kyoto university had supported in this study, and also
thank for emeritus Prof. Haruo Hisazumi, Department ofUrology, Kanazawa University.
REFERENCES:[1] N. Miyoshi, A. B. Bibin, et al., "in hoe of going overthe present Clinical PD and PDT", Eds. by N. Miyoshi and R. H. Pottier, Sankeisha, Nagoya, ISBN978-4-88361-883-5 (2011) pp.33-39.
[2] N. Miyoshi, K. Kume, et al., International Proceedings, Ed. by G. Kostron, Medimond, Italy (2011)
pp.121-125.
[3] N. Miyoshi, K. Kume, et al., "Aminolevulinic acid"
Eds. by I. Okura and T. Tanaka, ISBN978-47954-0229-4, Tokyo Inst. Tech. Press, Tokyo (2011)
pp. 23-29.
[4] N. Miyoshi, T. Ogasawara, et al., "Recent Progress ofClinical and Basic Research of ALA" Eds. by N.
Miyoshi and S. Kaneko, Kashiwaba Neurosurgery
Hospital, Sapporo (2005) pp. 11-16.

腫瘍特異性を増させた捕捉剤投与による BNCT と光線力学治療の共同通常
併用療法による前臨床共同研究（福井大学・医）三好憲雄、Andriana B. Bibin（筑波大学・医系）山本哲哉、松村明（京大・原子炉）田中浩基、小野公二 (A. N. Nesmeryanov Institute of Organoelement Compounds, Moscow) V. N.
Kalinin, V. A. Ol'shevskaya, A. V. Zaitsev (Blokhin National Cancer Center, Moscow) A. A. Shtil