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Nuclear Medicine and Biology 37 (2010) 903 – 910
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Radioiodinated 4-iodo-L-meta-tyrosine, a system L selective artificial
amino acid: molecular design and transport characterization in Chinese
hamster ovary cells (CHO-K1 cells)
Naoto Shikanoa,⁎, Takashi Kotania , Syuichi Nakajimaa , Masato Oguraa , Shinya Nakazawaa ,Jun-ichi Sagarab , Masato Kobayashic , Takeshi Babad , Naoto Yamaguchid ,Nobuo Kubotaa , Keiichi Kawaic
aDepartment of Radiological Sciences, Ibaraki Prefectural University of Health Sciences, 4669-2 Ami, Ami-machi, Inashiki-gun, Ibaraki 300-0394, Japan
Center for Humanities and Sciences, Ibaraki Prefectural University of Health Sciences, 4669-2 Ami, Ami-machi, Inashiki-gun, Ibaraki 300-0394, Japanc
Division of Health Science, Graduate School of Health Sciences, Kanazawa University, 5-11-80 Kodatsuno, Kanazawa, Ishikawa 9200-942, JapandCenter for Medical Science, Ibaraki Prefectural University of Health Sciences, 4669-2 Ami, Ami-machi, Inashiki-gun, Ibaraki 300-0394, Japan
Received 30 March 2010; received in revised form 28 April 2010; accepted 3 June 2010b

Abstract
Introduction: High expression of the system L amino acid transporter has been observed in clinically important tissues including tumors andthe blood-brain barrier. We examined amino acid transport system L selectivity of 14C(U)-L-tyrosine (14C-Tyr), 125I-4-iodo-L-meta-tyrosine(4-125I-mTyr), 125I-6-iodo-L-meta-tyrosine (6-125I-mTyr), 125I-3-iodo-α-methyl-L-tyrosine (125I-IMT) and 125I-3-iodo-L-tyrosine (3-125I-Tyr)using Chinese hamster ovary cells (CHO-K1).
Methods: Cells in the exponential growth phase were incubated with 18.5 kBq of labeled amino acid in 2 mL of phosphate-buffered salinebased uptake solution and an uptake solution with/without Na+ at 37°C or 4°C. We examined the effects of the following compounds (1.0mM) on transport: 2-(methylamino)isobutyric acid (a specific inhibitor of system A, in Na+-containing uptake solution); 2-amino-bicyclo[2,2,1]heptane-2-carboxylic acid (a specific inhibitor of system L, in Na+-free uptake solution); sodium azide and 2,4-dinitrophenol (NaN3and DNP, inhibitors of the generation of adenosine triphosphate); p-aminohippurate and tetraethylammonium (PAH and TEA, inhibitors oforganic anion and cation transporters); and L- and D-isomers of natural amino acids.
Results: 14C-Tyr exhibited affinity for systems L, A and ASC. 4-125I-mTyr and 3-125I-Tyr exhibited high specificity for system L, whereas
6-125I-mTyr and 125I-IMT exhibited affinity for both systems L and ASC. Uptake of 4-125I-mTyr was markedly reduced by incubation at 4
°C, and was not significantly inhibited by NaN3, DNP, PAH or TEA. The inhibition profiles of the L- and D-isomers of natural amino acids
indicated that system L mediates the transport of 4-125I-mTyr.
Conclusions: 4-125I-mTyr exhibited the greatest system L specificity (93.46±0.13%) of all of the tested amino acids.
© 2010 Elsevier Inc. All rights reserved.
Key words: 4-iodo-L-meta-tyrosine; System L; Neutral amino acid; Chinese hamster ovary cells

1. Introduction
Amino acid uptake by cells appears to be one of the mostimportant processes involved in cellular functions. System L
is an important neutral amino acid transport system, because
it is a major route by which cells are supplied with large⁎ Corresponding author. Tel.: +81 298 40 2217; fax: +81 298 40 2317.
E-mail address: sikano@ipu.ac.jp (N. Shikano).
0969-8051/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.nucmedbio.2010.06.006

neutral amino acids, including branched and aromatic aminoacids. Substrates of system L include some essential aminoacids and precursors of neurotransmitters [1]. System L
transporters are expressed at high levels in angioendothelial
cells of the blood-brain barrier and the placenta barrier [2].
Alterations in amino acid transport systems in the brain are
frequently associated with neuropsychiatric disorders, suchas Parkinson's disease [3] and schizophrenia [4]. Also,
system L is up-regulated in many kinds of tumor cells undergoing active growth and proliferation [2]. Because of its

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broad substrate selectivity, it has been proposed that system L
transports not only naturally occurring amino acids but alsoamino-acid related compounds such as L-DOPA (therapeutic
drug for Parkinson's disease), meluphalan (anticancer
phenylalanine mustard), triiodothyronine (thyroid hormone),
thyroxine (thyroid hormone), gabapentin (anticonvulsant),
and S-(1,2-dichlorovinyl)-L-cysteine (neurotoxic cysteine
conjugate) [5–10]. System L plays important roles in themetabolism and pharmacokinetics of the abovementioned
drugs and compounds. Thus, the available evidence suggeststhat system-L-specific artificial amino acids would be usefulas imaging agents for studies of tumors, cerebral functions
and other important biological phenomena.
Systems L, A and ASC, which are the neutral amino acidtransport systems of the plasma membrane, have different
biochemical characteristics. System L is defined by its Na+independency and inhibition by 2-amino-bicyclo[2,2,1]
heptane-2-carboxylic acid (BCH). System A is defined by
its Na+ -dependency and inhibition by 2-(methylamino)
isobutyric acid (MeAIB). System ASC is a Na+-dependenttransport system that is intolerant of N-methylation ofsubstrates, but no specific substrate for system ASC has yet
been found. These systems have been characterized in astudy by Shotwell et al. using Chinese hamster ovary cells[11]. A well-characterized, popular cell line such as CHO-K1
may be a useful tool for the development of artificial aminoacids, by facilitating comparisons of transport properties
between different amino acid transport markers.
System L1 and L2 (components of system L) genes areclassified into two different families of solute carriers (SLCs),
the SLC7 and SLC43 families, respectively, based on thestandards established by the Human Gene Nomenclature
Committee [12–15]. The SLC7 family is further divided into
the cationic amino acid transporter family and the L-typeamino acid transporter (LAT) family [12–14]. The two
known isoforms of system L1 belong to the LAT family andare named type 1 (LAT1) and type 2 (LAT2). The LAT1 andLAT2 transporters of system L1, but not the LAT3 and LAT4transporters of system L2, form a heterodimeric complex

with an additional protein, the heavy chain of the 4F2
antigen (CD98; 4F2hc/SLC3A2), which is required for
LAT1/2 transport function. LAT1/2 and LAT 3/4 also differ
in that LAT1 and LAT2 selectively transport neutral aminoacids by obligatory exchange mechanisms, whereas LAT3
and LAT 4 did not display obligatory exchange mechanisms. Although these transporters show partly overlapping
expression patterns and substrates, some differences in theirexpression and substrates have been observed [12–15].
Since tumors require increased uptake of amino acids fortheir enhanced growth and proliferation, it is of interest that
LAT1 is strongly up-regulated in many cancer cells both invitro and in vivo [16].
The amino acid Tyr is important biologically, not only as
a component of proteins and a precursor of thyroid hormones
but also (in its hydroxylated form dopa) as a precursor ofcatecholamines and melanin [17]. Previous findings suggested that Tyr derivatives could be used to measure neutralamino acid transport.
We previously investigated the biodistribution andexcretion of the following compounds in mice, using metabolite analysis: 125I-4-iodo-L-meta-tyrosine (4-125 I-mTyr),125
I-6-iodo-L-meta-tyrosine (6-125 I-mTyr), 125 I-3-iodo-αmethyl-L-tyrosine (125 I-IMT), naturally occurring 14C(U)-Ltyrosine (14C-Tyr), and 125I-3-iodo-L-tyrosine (3-125I-Tyr)(Fig. 1). We have presented our findings regarding the invivo pharmacological features of those compounds in several
reports [18–21]. The artificial amino acid 4-125I-mTyrexhibited high metabolic stability, rapid blood clearance,
rapid urinary excretion and a biodistribution similar to that of
125
I-IMT [21]. However, the pharmacological characteristicsof 4-125 I-mTyr differed greatly from those of 125I-IMT,
particularly in renal handling [21].
Mechanisms of accumulation of the above compounds(except for 125 I-IMT) have not been fully clarified. In thepresent study, using CHO-K1 cells and a modification of themethod of Shotwell et al., we examined the mechanisms of
uptake of iodinated tyrosine analogues, with particularemphasis on 4-125 I-mTyr. The present findings should be

Fig. 1. Chemical structures of 14C(U)-L-tyrosine (14C-Tyr), 125I-4-iodo-L-meta-tyrosine (4-125I-mTyr), 125I-6-iodo-L-meta-tyrosine (6-125I-mTyr), 125I-3-iodo-Ltyrosine (3-125I-Tyr) and 125I-3-iodo-α-methyl-L-tyrosine (125I-IMT).

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useful for the development of radiopharmaceuticals thattarget system L amino acid transport [16].

The growth curve was plotted based on growth up to 6 days
after inoculation. Cells were used for experiments on thethird or fourth day after inoculation.

2. Materials and methods

2.4. Time course of uptake of labeled amino acids

2.1. Materials

After removal of the culture medium, each of the above
60-mm dishes was washed once with 5 mL of incubation
medium for 10 min at 37°C. The incubation medium
consisted of 137 mM NaCl, 2.7 mM KCl, 8.0 mM
Na2HPO4·12H2O, 1.5 mM KH2PO4, 1.8 mM CaCl2·2H2O,
1.0 mM MgCl·6H2O and 5 mM 2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid, with a final pH of 7.4. The cells werethen incubated with 2 mL of incubation medium containing
18.5 kBq of labeled amino acids, for 5, 10, 15 or 30 min at
37°C. The incubation medium was then aspirated, and thecell monolayers were rapidly rinsed twice using 5 mL of icecold incubation medium. The cells were then solubilized in
1.5 mL of 1 N NaOH, and the radioactivity of each aliquot
was counted. We measured the radioactivity associated withsolubilized cells using an ARC-380 well-type scintillation
counter (Aloka) for 125I-labeled compounds, and using an
LS6500 liquid scintillation counter (Beckman Instruments,
Fullerton, CA, USA) for 14C-labeled compounds.

The radiolabeled precursors (D,L-mTyr, α-methyl-Ltyrosine and L-tyrosine) were purchased from SigmaAldrich Japan Co. 125I-NaI was purchased from Amersham
Pharmacia Biotech. 14C-Tyr was purchased from American
Radiolabeled Chemicals Inc. Chinese hamster ovary cells(CHO-K1) were purchased from the RIKEN Bioresource
center cell bank. Plastic tissue culture dishes (diameter,
60 mm) were purchased from Falcon. Plastic culture flasks(surface area, 25 cm2) were purchased from Nalge Nunc
International. 2-amino-bicyclo[2,2,1]heptane-2-carboxylic
acid (BCH, a system-L-specific inhibitor), 2-(methylamino)
isobutyric acid (MeAIB, a system-A-specific inhibitor),natural L/D-amino acids and other chemicals of reagent
grade were purchased from Kanto Chemical.
2.2. Labeled Amino acids
125I-3-iodo-α-methyl-L-tyrosine (125 I-IMT) and 125I-3iodo-L-tyrosine (3-125I-Tyr) were prepared using conventional methods described elsewhere [18–21]. The methods
used to prepare the other labeled amino acids are describedin detail elsewhere, and are described here briefly. For 125I4-iodo-L-meta-tyrosine (4-125 I-mTyr) and 125 I-6-iodo-Lmeta-tyrosine (6-125 I-mTyr), we separated L-mTyr from D,
L -mTyr by high-performance liquid chromatography(HPLC) using a Crownpak CR(−) chiral column (Daicel
Chemical Industries). The separated L-mTyr was labeledwith 125I-NaI and Chloramine-T (Kanto Chemical). Radioiodination of L-mTyr produces two major geometricisomers: 6-iodo- and 4-iodo-L-mTyr [20]. To divide andpurify those two isomers, we performed HPLC with a NovaPak C18 column (Waters). Radiochemical yield waschecked using a silica gel thin-layer chromatography kit(Merck). The labeled compounds were used in their noncarrier-added condition.
2.3. Cell line
We performed the cellular uptake experiment using amodification of the method of Shotwell et al. [11]. CHO-K1
cells were maintained by serial passage in 25-cm2 cell
culture flasks. The cells were fed with Dulbecco's modified
Eagle's medium (Sigma-Aldrich Japan K.K.) supplemented
with L-glutamine (2 mM) and 10% fetal bovine serum
without antibiotics, in an atmosphere of 5% CO2 and 95% air
at 37°C (pH 7.4). Subculture was performed every 5 days
using 0.02% ethylenediamine tetra-acetic acid (EDTA) and
0.05% trypsin.
CHO-K1 cells were seeded on 60-mm dishes at a densityof 5×105 cells/cm2 in 5.0 ml of the complete culture medium.

2.5. Determination of transport system selectivity of labeledamino acids
We examined the system-L specificity of transport of thelabeled amino acids in CHO-K1 cells. One of the following
compounds was added to cell cultures at a final concentrationof 1 mM: BCH in incubation medium without Na+; or
MeAIB in incubation medium with Na+. The cells werethen incubated in 2 mL of incubation medium with/without
Na+ containing 18.5 kBq of one of the labeled amino acidsfor 5 min at 37°C. In the medium without Na+, NaCl was
replaced with the same concentration of choline-Cl. The
other experimental conditions were the same as those of thetime course experiment.
2.6. Characterization of 4-125I-mTyr transport
For the inhibition experiment, we added one of thefollowing compounds (final concentration, 1.0 mM) to cell
cultures in incubation medium containing Na+ and 18.5 kBqof 4-125 I-mTyr [22]: p-aminohippurate (PAH, inhibitor oforganic anion transporter), tetramethylammonium (TEA,
inhibitor of organic cation transporter), 2,4-dinitrophenol(DNP, inhibitor of adenosine triphosphate generation) or
sodium azide (NaN3, inhibitor of adenosine triphosphate
generation). Because it has previously been demonstrated
that both enantiomers of amino acids are endogenously
present in mammalian tissues [23,24], we tested the transportinhibitory properties of both D- and L-amino acids, at a final
concentration of 1.0 mM. The cells were incubated with theabove compounds for 5 min at 37°C. To examine effectsof low temperature, we incubated cells for 5 min at 4°C.

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2.7. Statistical analysis
Data were collated as the mean±S.D. of three to fivemeasurements, and each experiment was performed in
duplicate. Results were analyzed using Student's t test.
Probability levels of Pb.0001 were considered to indicate
statistical significance.

3. Results
Fig. 2 shows the growth curve of the CHO-K1 cells. It
took 5 days for the cells to reach confluence. On the second
to fourth day after inoculation, the cells were in the
exponential growth phase. Cells were used for experimentson the third or fourth day after inoculation.
In the time course analysis of uptake of labeled aminoacids into CHO-K1 cells, steady-state levels were reachedabout 10 min after the start of incubation (Fig. 3). An
incubation time of 5 min was used for all uptake
measurements. Mother 14C-Tyr had the highest rate of
uptake at steady-state (Fig. 3A). Uptake of 4-125I-mTyr wasmuch greater than that of 6-125I-mTyr or 3-125I-Tyr for up to
15 min after the start of incubation (Fig. 3B).
Fig. 4 shows the contributions of individual transportsystems to total uptake of the 5 labeled amino acids. Systems
A, ASC and L mediated uptake of mother 14C-Tyr. Systems
ASC and L mediated uptake of 125I-IMT and 6-125I-mTyr.
The labeled amino acids 4-125 I-mTyr and 3-125 I-Tyr
exhibited high specificity for Na+ -independent uptake
mediated by system L.
Uptake of 4-125 I-mTyr was markedly reduced byincubation at 4 °C (3.3%). None of the chemical inhibitors
significantly inhibited uptake of 4-125 I-mTyr (Fig. 5).
Neutral amino acids (Ala, Ser, Thr, Cys, Leu, Ile, Phe,
Met, Tyr, His, Trp Val and Asn) inhibited uptake of 4-125ImTyr (Fig. 6). However, D-amino acids (Ala, Ser, Thr andCys) had weak or statistically insignificant inhibitory effects
on uptake of 4-125I-mTyr. L-isomers had stronger inhibitory
effects than D-isomers.

Fig. 2. Growth curve of CHO-K1 cells.

Fig. 3. Time course analysis of uptake of 14C-Tyr (A) and the 125I-labeled
tyrosine analogues (B) into CHO-K1 cells. Each bar represents the mean±
S.D. of five monolayers.

4. Discussion
In experiments using Chinese hamster ovary cells,
Shotwell et al. have determined the individual contributionsof transport systems L, A and ASC to the total uptake ofnaturally occurring neutral amino acids [11]. They reported
that most neutral amino acids enter cells via multiple
transport systems. System L was named for its “leucinepreferring” characteristics, although it also transports otherlarge neutral amino acids [15]. In a review article,
Christensen concluded that leucine and phenylalanine should
not be considered specific substrates of system L [1]. BCH,
which is a non-metabolizable amino acid analogue, iscommonly used (in place of naturally occurring neutralamino acids) as a specific model compound for system L
transport in uptake solution without Na+ [1].
Radioiodination has been extensively used to label
compounds of medical/biologic interest. However, thechemical structure of BCH suggests that it is difficult toradiolabel, especially with a single photon emission nuclide.
Tyr derivatives labeled with a γ-emitter (125I or 123I) or apositron emitter (124I) are suitable radiopharmaceuticals forstudies of system L transport specificity. The phenol ring is aconvenient site for labeling of Tyr and its derivatives

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907

Fig. 4. Experimental definition and comparison of contributions of neutral amino acid transport systems to uptake of 14C-Tyr (A), 4-125I-mTyr (B), 6-125I-mTyr(C), 3-125I-Tyr (D) and 125I-IMT (E) into CHO-K1 cells. Each bar represents the mean±S.D. of five monolayers. ⁎Pb.0001; NS, not significant.

(Fig. 1). In the present study, we prepared 125 I-labeled
compounds by performing direct electrophilic radioiodination using the Chloramine-T method, because it is an easy
way to prepare radioiodinated compounds [20].
In the present study, transport of mother 14C-Tyr was
mediated by systems A, ASC and L, and 14C-Tyr is therefore
not a specific substrate of a single neutral amino acidtransport system in CHO-K1 cells (Fig. 4). We speculated
that the increase in weight or bulkiness caused byintroduction of iodine into the phenol ring of Tyr couldchange the affinity of Tyr for these amino acid transportsystems. The molecular weight of Tyr (or mTyr) and αmethyl tyrosine is about 181 and 195, respectively, and theatomic weight of the stable isotope of iodine is about 127.

Thus, the atomic weight of iodine accounts for approximately 41% and 38% of the molecular weight of C9H11NO3I(for 3-I-Tyr, 4-I-mTyr and 6-I-mTyr) and C10H13NO3I (forIMT), respectively. The present results indicate that thesemolecular alterations caused changes in their specificity foramino acid transport systems (Fig. 4). Also, differences in
the position of the iodine and hydroxyl group in the phenol
ring and in the presence of an alpha-methyl group caused
differences in specificity for amino acid systems.
In the present study, mono-iodination of the phenol ringcaused a lack of affinity for system A and an increase in
specificity for system L (Fig. 4B–E), compared to 14C-Tyr(Fig. 4A). For example, 3-125I-Tyr exhibited no affinity forsystem A transport.

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Fig. 5. Effects of inhibitors on accumulation of 4-125I-mTyr in CHO-K1 cells
incubated in uptake solution with Na+. Each column represents the mean±S.
D. of five monolayers. *Pb.0001.

The position of iodine in the phenol ring appears to be an
important factor in the system selectivity of radioiodinatedtyrosine derivatives. In the present study, 3-125I-Tyr and
4-125 I-mTyr exhibited high specificity for system L transport(Fig. 4 B and D), whereas 6-125I-mTyr exhibited affinity forboth system L and system ASC (Fig. 4C). Mono-iodination
at the meta/para position of the phenol ring resulted in
specificity for system L. Mono-iodination at the ortho
position of the ring resulted in affinity for systems ASC and
L. Also, alpha-methylation of 3-125I-Tyr conferred affinity
for system ASC, as indicated by the results for alphamethylated 3-125I-Tyr (IMT) (Fig. 4 D and E).
We further examined in vitro transport of 4-125I-mTyr,
which exhibited the greatest selectivity for system L. PAH
and TEA did not inhibit uptake of 4-125I-mTyr into CHO-K1
cells, indicating that transport of 4-125I-mTyr was not
mediated by organic anion or cation transporters. DNP and

Fig. 6. The effects of L- and D-amino acids on uptake of 4-125I-mTyr into CHO-K1 cells.

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125

NaN3 did not inhibit uptake of 4- I-mTyr, indicating that
uptake of 4-125I-mTyr did not involve active transport.
However, uptake of 4-125I-mTyr was markedly reduced byincubation at 4°C, indicating that transport of 4-125I-mTyr
was carrier-mediated (Pb.0001). There was no significant
difference in uptake of 4-125I-mTyr between uptake solutions
with and without Na+, indicating that transport of 4-125ImTyr was Na+-independent (Fig. 4B). Because previous
studies indicate that D-amino acids are endogenously present
in mammalian tissues [23,24], we used both D- and L-aminoacids in the present inhibition experiment. Neutral D- and125
L-amino acids inhibited uptake of 4I-mTyr (Fig. 6).
The inhibition profiles in Fig. 6 are similar to those of theL-type amino acid transporter 1 (LAT1), which is a memberof the system L transporter family. A similar inhibition
profile has previously been obtained using the porcine
kidney epithelial cell line LLC-PK1, which expresses LAT1
[25–27]. Thus, the present results indicate that uptake of
4-125 I-mTyr is mediated by a Na+-independent neutralamino acid transporter, most likely LAT1 (which is aneutral amino acid exchanger).
We believe there is a need for novel radiopharmaceuticals
that are useful for assessment of system L amino acidtransport function in clinical studies. Targeting of system L
transport in tumor cells would greatly facilitate imaging oftumors, cerebral functions and other important biologicalphenomena in vivo.
To produce a useful radioiodinated in vivo radiotracer,
the radioiodine must be attached in such a way that theresultant radiotracer has affinity for system L transport andin vivo stability. It is important that the radioiodinatedcompound is not broken down quickly via general metabolic pathways, because a complicated metabolic fate ofa radioiodinated compound would require a complicatedkinetic model for quantitative study [28]. Of the fivetyrosine derivatives used in the present study, 125I-IMT,
4-125 I-mTyr and 6-125I-mTyr display metabolic stability,
rapid blood clearance, rapid urinary excretion and rapid
biodistribution, and are excreted as intact amino acids[19–21]. In contrast, natural 14C-Tyr and 3-125I-Tyr are notstable in vivo, and they have complex metabolic fates [18].
Stability against deiodination is essential, in order to prevent
internal irradiation of organs that accumulate radioiodine,such as the thyroid and stomach.
In conclusion, 4-125 I-mTyr shows promise as a novel
system-L-selective amino acid transport marker. Further
investigation is needed to develop methods for using 4-125ImTyr in SPECT, and to clarify the behavior of 4-125 I-mTyr ineach target organ and tissue.
We examined the system L specificity of transport ofiodinated tyrosine derivatives in CHO-K1 cells. Differencesin the position of the iodine and hydroxyl group in the phenol
ring and in the presence of an alpha-methyl group caused
differences in the amino acid transport system selectivity ofthe tyrosine derivatives. Transport of mother 14C-Tyr was
mediated by systems A, ASC and L. 4-125I-mTyr and 3-125I-

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125

Tyr exhibited high specificity for system L, whereas 6- ImTyr and 125 I-IMT exhibited affinity for both system L andsystem ASC. Of the 5 tyrosine derivatives used in the present
study, 4-125 I-mTyr appears to be most suitable for imaging ofsystem L transport. Further study is needed to examinetransport of 4-125I-mTyr via other transport systems.

Acknowledgments
We would like to thank Yusuke Kishi, Misako Tanaka,
Asami Teraoka, Junichi Ohyama, Sae Fujimoto, Yuki
Nobayashi and Ayako Yagi (Ibaraki Prefectural University)
for their technical assistance. This work was supported by
Grants-in-Aid for Scientific Research (#10770451,#14770498, #13557075, #15659283, #16659322,#17390336 and #22591372) from the Ministry of Education,
Science, Sports and Culture of Japan and the Japan Society for
the Promotion of Science. Financial support was also provided
by the Ibaraki Prefectural University Research Project (9808,
0118, 0220, and 0944) and Ibaraki Prefectural University
Grants-in-Aid of the Encouragement for Young Scientists
2001, 2002, 2004, 2005, 2006, 2008, 2009 and 2010.

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