CO6-1

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

K. Akamatsu, N. Shikazono and T. Saito1

r 

Irreparable DNA Damage Analysis Research Group,
Quantum Beam Science Research Center, Japan Atomic
Energy Agency (JAEA)
1
Research Reactor Institute, Kyoto University
INTRODUCTION:
DNA lesions induced by ionizing radiation andchemicals can cause mutation and carcinogenesis. In
particular, “clustered damage” site, that is a DNA region
with multiple lesions within one or two helical turns, isbelieved to hardly be repaired. This damage is considered
to be induced, e.g., around high-LET ionizing radiation
tracks. However, detail of the damage is not known. We
have already developed a method for estimating degreeof localization of abasic sites (APs) in DNA using Förster
resonance energy transfer occurred between different
fluorescence probes (“hetero-FRET” using Alexa350 andAlexa488) [1] . The results showed that 12C5+ beam produced close APs within a track: the apparent distancecalculated was approximately 17 base pairs [2]. Thisfinding indicates that direct radiation effect of 12C5+ beamnear the Bragg peak produces clustered DNA damage.
We have recently applied the method to DNA in acell-mimetic radical scavenging condition [3,4]. However,there are some problems of the complex protocol and of
the sensitivity due to the low extinction coefficient of
Alexa350. We have, therefore, developed “homo-FRET”
occurred between two or more Alexa488 molecules. We
will obtain magnitude of FRET also from “fluorescenceanisotropy” of homo-FRET between Alexa488 molecules.
The new protocol using homo-FRET enables us to estimate DNA damage localization without any enzymes andimproves sensitivity to detect a clustered damage.

IVV  G IVH ,IVV  2G IVH

where IVV is the fluorescence intensity when the excitation and emission polarizers are both vertically oriented.
IVH is one when the excitation/emission polarizers arevertically/horizontally oriented. G is the grating factor
defined as IHV/IHH.
RESULTS AND DISCUSSION:

Fig. 1. Relationship between AP average density (the numberof APs per base pair) and fluorescence anisotropy for 60Co
-rays (●). The solid line indicates a theoretical curve for Poisson distributed APs in DNA.

EXPERIMENTS:●Sample preparation and -irradiation
The plasmid DNA digested by Sma I was used (linear
form). The DNA was dissolved to be 0.1 g/L in 0.2 M
Tris-HCl buffer (pH 7.5) which is a cell-mimetic condition in relation to radical scavenging capacity. Twenty
microliters of the DNA solution was transferred to a microtube (0.5-mL size), and was irradiated with 60Co
-rays (LET: ~0.2 keV/m; Kyoto University Research
Reactor Institute: KURRI) as a standard radiation source.

In general, fluorescence anisotropy decreases withincreasing FRET [5]. As shown in Fig.1, there was a little difference between the -ray data points and a theoretical curve based on Poisson distribution. This finding
suggest that APs produced by the -rays are likely to belocalized compared to those randomly distributed. Thistendency is similar to the hetero-FRET results as shown
previously [2-4]. A radiation “spur” on or nearby DNA
might sometimes produces clustered damage. In any
case, knowledge of AP distribution for 60Co -rays as areference is quite important to study DNA damage by
ionizing radiation.
REFERENCES:[1] K. Akamatsu, N. Shikazono, Anal. Biochem. 433(2013) 171-180.
[2] K. Akamatsu, N. Shikazono, and T. Saito, Radiat.
Res. 183 (2015) 105-113.
[3] K. Akamatsu, N. Shikazono, and T. Saito, KURRI
Progress Report 2012 (2013) 263.
[4] K. Akamatsu, N. Shikazono, and T. Saito, KURRI
Progress Report 2014 (2014) 127.
[5] L. W. Runnels, and S. F. Scarlata, Biophys. J. 69(1995) 1569.

●Preparation of fluorophore-labeled irradiated DNA andthe FRET observation
The irradiated DNA (10 L in water) and 10 L of
100 mM Tris-HCl (pH 7.5) were mixed in a microtube.
Two microliters of Alexa488/DMSO was added to the
DNA solution and was incubated for 24 h at 35°C. The
fluorophore-labeled DNA was purified by ethanol-precipitation followed by ultrafiltration. The fluorescence anisotropy was measured at 525 nm (ex. 470 nm).
The anisotropy, < r >, is defined as follows:

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CO6-2

Structural Insights of the Degradation Scheme of Yeast 20S Proteasome
Studied by Genetic Mutation and AFM Motion Imaging

H.Yamaguchi1,T.Amako1, M.Unno2, I.Hisatome3,
K.Hosokawa1 and Y.Morimoto

mechanism. AFM images of the 26S proteasome were
measured by NanoExplorer at 20°C at the Research Institute of Biomolecule Metrology in Tsukuba. Particle
analysis was carried out by AFM reconstitution.

Research Reactor Institute, Kyoto University
School of Science and Technology,
Kwansei Gakuin University
2
Graduate School of Science and Engineering,
Ibaraki University
3
Institute of Regenerative Medicine and Biofunction,
Tottori University
1

INTRODUCTION: Proteasomes are the multicatalytic
protein complexes with large molecular weight. It is wellknown that the ubiquitin proteasome system plays animportant role in regulated proteolysis. Proteasome works
as 26S particle that is composed of two 19S regulating
components and a 20S proteasome. The 20S proteasome
forms barrel shape and is composed of four rings, α- andβ-rings; each ring contains highly homological seven αand seven β-subunits. Each of 1, 2 and 5 β-subunit has
different enzyme activity; 1 is caspase-, 2 tryptic- and 5
chymotryptic- like activity, respectively. It was foundrecently that inhibition of 20S proteasome activity resulted in decrease and disappearance of cancer cells. The
complex structure of 20S proteasome and its inhibitor
have been determined by X-ray diffraction method.
Binding site of inhibitor is closed to Tyr170 and Thr1 ofthe β5 subunit. It is considered that an aromatic ring of
the inhibitor interacts with Y170 by ring-stacking force.
Since weak interaction of compounds to active aminoacid residues we found, processing or degradation assay
have been clarified by use of genetic mutations of theyeast 20S proteasome. Tyrosine mutations to Ala, Pheand Leu may not have interaction with the inhibitor andstrictly changes binding activities to the 19S particle. The
whole particle, 26S proteasome, including such 20S coreis very important in the actual living cell for a degradation or stabilization on the cell circulation. Especially, a
motion or behavior of the 26S proteasome when a regulatory particles bound should be clarified at atomic- or
nano-scale resolution, we have tried to visualize such
behavior as a motion capture of the atomic force microscopy (AFM) technique.
EXPERIMENTS: Yeast 20S (wild, and genetic modifications in the β5 subunit; Y170A, Y170L and Y170F)
and 26S proteasome particles were prepared by using
yeast (Saccharomyces cerevisiae) cell cultivated. The
crude extract was purified by using M2 affinity andMono-Q anion exchange chromatography techniques.
When the 26S particle was isolated, gel filtration chromatography was continuously applied with a buffer solution including ATP. Isolated 20S proteasome was concentrated by ultrafiltration. Activity measurement of wild
and mutant proteasomes in the presence of the inhibitor
was carried out in order to reinvestigate the inhibition

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RESULTS: Genetic mutation for Y170 would haveactivities because of a lack of aromatic ring in the aminoacid residue in the Ala and Leu, binding ability of theinhibitor might be lost. Typical activity assay forwild-type 20S shows reduction, otherwise the Y170L has
no decrease of activity assay. Since additive order or
combination of substrates and inhibitors affects hydrolysis strongly, 26S whole particle may have specific character in terms of own-shape.
The AFM measurement shows whole
structure of the 26S particle, and analyses of resultant
images describe a shape and size, and also a structural
motion. Intensity distribution analysis, which reconstitutes outer shape of particle by enormous amount of omnidirectional image data, was applied for the AFM image
that is able to judge as 26S proteasome. Intensity distribution of AFM data (Figure 1a) shows symmetrical shape,
which is consistent with the size and shape of a double-capped whole 26S particle composed of central 20S
and the both ends (Figure 1b). Measurements of AFM
images of proteasome in the presence of substrates are in
progress in order to analyze dynamic motion when proteasome is working.
a)b)

Figure 1. Distribution analysis of AFM data of 26S
proteasome.
a) Intensity distribution of 26S particle of AFM image.
b) Molecular model of 26S proteasome particle.

CO6-3The Application of Cancer Immunotherapy for the combination with
Boron Neutron Capture Therapy (BNCT)

T. Suzuki, K. Horie, R. Abe, S. Masunaga1, N. Kondo1,
Y. Sakurai1, M. Suzuki1 and K. Ono1

administration of mAb could not lead to significant anti-tumor effect during combination therapy, suggestingthat the appropriate timing of mAb treatments is important for the induction of strong anti-tumor effect during the combination therapy. Based on these observation, now, we are trying to establish the effective combination therapy with BNCT and immunotherapy.

Recerch Institute of Biomedical Sciences,
Tokyo University of Science
1
Research Reactor Institute, Kyoto University
Introduction
Boron neutron capture therapy (BNCT) is an attractive
therapy for local tumor control in the treatment of brain
tumor, melanoma, and so on [1,2]. However, some important issues are remained: the tumor-specific accumulation of highly concentrated boron, the real-time quantification of boron concentration in local tumor tissues, andthe prevention of re-growth and metastasis of residual
tumor cells after BNCT. Recently, much progress has
been seen in tumor immunotherapy, and attracts many
attentions of clinical researchers [3,4]. To achieve successful tumor regression, we try to establish the combination treatments with BNCT and immunotherapy. Using
the radiotherapy with X ray irradiation, we firstly examined the anti-tumor effect of the combination therapy.
Results and Discussions
The establishments of cancer immunotherapy
In last year, we established some immunotherapies,
e.g. blockade of immunological checkpoints with anti
PD-1mAb and/or anti CTLA-4 mAb, vaccination withdendritic cells (DCs), and adoptive cell transfer therapy(ACT), using mouse model. Until now, we investigatedthe mechanisms of potent anti-tumor immune response
by ACT under lymphopenic condition [5,6]. Assessing
anti-tumor effect by anti PD-1 mAb or anti CTLA4 mAb,
we confirmed the anti-tumor effect by blockade of immunological check points (Fig.1). In addition, similar toclinical trials, we found that anti-tumor effect was not
sufficient for the tumor regression in some mice, whereas
few mice treated with mAb could reject tumors. Using
this protocols, we assessed whether anti-tumor effect isenhanced by the combination with radiotherapy.
Anti-tumor effect by the combination with radiotherapy and immunotherapy.
To assess the effect of combination therapy with radiotherapy and immunotherapy, tumor bearing mice weretreated by mAb, and were locally irradiated by X ray(12Gy). Expectedly, significant tumor regression wasobserved in mice treated with the combination therapy(Fig.2). Analyzing anti-tumor immune response, we
found that the induction of tumor antigen specific CTLs
was not augmented by combination therapy, whereas
local X ray irradiation lead to the increase of CTL population. Notably, compared with prior-treatment, later

REFERENCES:[1] M. Suzuki, et al. Int. J. Radiat. Oncol. Biol.
Phys. 58(3): 892-896, 2004.
[2] M. Suzuki, et al. Int. J. Radiat. Oncol. Biol.
Phys. 60(3): 920-927, 2004.
[3] Lifu Dengu et al., J. C. I., 124 (2), 687, 2014.
[4] M. A. Curran et al., PNAS, 107(9), 4275, 2010.
[5] Suzuki T. et al, J. Immonol. 180:4596-4605,
2008[6] Suzuki T. et al, Cancer Immunol. Immun.
65(3): 341-345, 2016

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CO6-4Development of Experimental Techniques for Neutron Biology

T. Chatake, S. Fujiwara1, Y. Yanagisawa2 and I.
Tanaka3

further analysis of the hydration structure is in progress.

Research Reactor Institute, Kyoto University
1

National Institute for Quantum and Radiological Science and Technology
2
3

Department of Pharmacy, Chiba Institute of Science
Department of Technology, Ibaraki University

INTRODUCTION: Recent neutron source had achieved
rapidly advancing development in a quarter century. Especially, the pulsed neutron source at J-PARC had enabled us various neutron experiment, which could neverbeen carried out due to lower neutron beam from neutron
reactor. In the present project, the following the forefront
experiments were studied for neutron biology. (1) D/H
contrast neutron protein crystallography (Fujiwara) (2)The combination of scattering experiments of physiologically active substances derived from Bacillus subtilis
natto (Yanagisawa), and (3) Dynamic neutron polarization technique for neutron protein crystallography(Tanaka). Here, the results of (1) and (2) are reported.
(1) D/H contrast neutron protein crystallography
EXPERIMENTS: Bovine pancreatic ribonuclease A(Sigma) was used for this study. Crystallization was carried out in H2O-solvent crystallization solution, in thesimilar manner as reported earlier [1]. The obtained
H2O-solvent crystal (h-crystal) was converted to
D2O-solvent crystal (d-crystal) by vapor diffusion method. Neutron diffraction data sets both of d- and h- crystals
were collected using BIX-3 diffractometer at JRR-3M
nuclear reactor. So far, the contrast between D and H
atoms was calculated in reciprocal space [2]. On the other
hand, we designed another calculation method (real space
D/H contrast method), and developed software for real
space D/H contrast method. This method was applied forthe two neutron data sets of the d- and h- crystals at 1.8
Angstrom [3].
RESULTS: The real space D/H contrast maps provided
detailed pictures of the H/D exchangeable atoms in ribonuclease A and its hydration with two superior properties
to the previous D/H contrast method. (i) all of the observed amplitudes can be used to visualize the D/H contrast, while only the amplitude commonly observed inboth d- and h- crystals can be used in the previous method. (ii) real space D/H contrast map can be easily incorporated into standard neutron crystallography. These advantages contributed to interpretations of the 3D structureof this protein including hydrogen atoms with high accuracy.
Solvent region was also investigated using D/H contrast
map. Fig. 1 shows average density in the D/H contrast
map along the distance from protein surface, where thesurface is accessible surface area. The results strongly
suggested the 1st hydration shell present 1.4-1.6 Angstrom apart from the protein surface, and the density
shape likes a donut with the width of ~1.2 Angstrom. The

Fig. 1.

Density of solvent region in D/H contrast map.

(2) Combination of scattering experiments of physiologically active substance derived from Bacillus subtilis
natto
EXPERIMENTS: Bacillus subtilis natto secrets abundantly water soluble vitamin K2 (MK-7). The chemicaland structural compositions of MK-7 have not been determined, while MK-7 has been sold as supplements. Our
aim of this project is to elucidate structural compositionby the combination of scattering method (DLS, XANS,
and SANS). The scattering method requires MK-7 ofhigh quality and quantity. In this physical year, procedureof purification was improved to achieve purification of
MK-7 with the highest quality ever.
RESULTS: 20 mM Tris-HCl (pH7.6) buffer was usedthrough purification. 1 L of Cultured medium was filteredand concentrated to 17.2 mL volume containing 0.3 M
NaCl before purification as described before [4]. 1st purification was carried out DEAE FF (5mL) (direct injection) with gradient of 0.3 M -> 1.0 M NaCl. The main
fraction was further purified using the 2 times of DEAE
FF (1mL) with the same gradient. After ion-exchange
chromatography, gel-filtration chromatography was carried out using sephacryl S-200 (φ16x60cm). At the firstgel-filtration, two peaks were observed in the elution
curve. The former peak was collected and, they were purified in the same gel-filtration again.
Finally, 891 uL solution of purified MK-7 could be obtained. UV280nm was 0.500. Bradford assay, in which BSA
was used as standard, indicated 0.087 mg/mL protein was
contained in MK-7. DLS and XANS are scheduled now.
REFERENCES:[1] Berisio, R., Lamzin, V. S., Sica, F., Wilson, K. S.,
Zagari, A. & Mazzarella, L. J. Mol. Biol. 292, 845–854(1999).
[2] Kossiakoff, A. A., Sintchak, M. D., Shpungin, J. &Presta, L. G. Proteins 12, 223–236 (1992).
[3] T. Chatake, S. Fujiwara, Acta Crystallogr. D72, 71-82(2016).
[4] Y. Yanagisawa, M. Sakanushi, M. Ishiyama, S.
Onuma, S. Fujiwara, T. Matsuo, T. Chatake, Y. Morimoto, KUR Progress Report 2013,P5-4 (2014).

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CO6-5

Design and Synthesis of Boron-containing Compounds for Effective Accumulaionin Tumor Tissues and Detection by Boron Magnetic Resonance Imaging

S. Aoki, Y. Nishiura, T. Tanaka, Y. Hisamatsu, Y. Sawamoto, and 2, induced by Cu(II) in aqueous solutions.
R. Araki1, T. Saido2, T. Suzuki3, K. Horie3, R. Abe3, S.
Masunaga4, K. Natsuko4, Y. Sakurai4 and K. Ono4
EXPERIMENTS and RESULTS:
Tumor accumulation of boronyl glucose compounds.
Faculty of Pharmaceutical Sciences, Tokyo University of
Low toxicity of the synthesized compounds were conSciencefirmed by animal tests. Although the distribution of1
these agents in tumor cells are low, it was found that T/B
Bruker Biospin K. K.
2ratios of some compounds are 2.3~2.4, which is better
RIKEN Brain Science Institute3
than that of BSH (0.5) [3].
Research Institute of Biomedical Sciences, Tokyo Uni11
versity of Science
B NMR and MRI change of carborane derivatives
4
by decomposition reaction promoted by copper(II).
Research Reactor Institute, Kyoto University
Decomposition reaction of 1 and 2, as well as our preINTRODUCTION:
Boron neutron capture therapy vious compounds [5] were followed by 11B NMR and(BNCT) is one of powerful therapies for local tumor con- MRI. Interestingly, the probe 2 undergoes faster detrol in the treatment of brain tumor, melanoma, and so on composition than that of 1 at 37 oC and neutral pH,[1]. Some critical issues are remained for the successful which was successfully detected on 11B NMR and MRI
BNCT, such as the tumor-specific accumulation of highly (Fig. 2) [4,6].
concentrated boron and the real-time detection of boron
concentrations in local tumor tissues. In this study, we
have designed and synthesized new boron compounds
appended with glucose moiety and metal chelators for
BNCT and B NMR (nuclear magnetic resonance) or MRI(magnetic resonance imaging) [2].
As for the boron compounds for more efficient accumulation in tumor tissues, we have synthesized several
glucose-based boron compounds and assessed their tumor/blood (T/B) ratios [3].
As for B-MRI,
some icosahedral carboranes
derivartives, 1 and 2, which are composed of 10 boron
atoms and 2 carbon atoms and has considerable thermaland chemical stability, were synthesized (Fig. 1). We
discovered the first example of full deboranation reaction ofo-carborane catalyzed by d-block metal ions under Fig 2. 11B MRI and 11B{1H} NMR (128 MHz) spectra ofphysiological conditions [4]. Among various d-block 2 (2 mM) in DMSO/0.5 M HEPES (pH 7)/D2O after inmetals tested, Cu2+ accelerates most efficiently to pro- cubation at different concentrations ((a)~(f) 0, 0.02, 0.1.
duce 10 B(OH)3 from one molecule of carborane 0.2 mM, 1.0, and 2.0 mM) of Cu(II).
deriva-tives.
REFERENCES:[1] a) R. F. Barth et al., Clin. Cancer Res., 11 (2005)
3987-4002. b) R. F. Barth et al. Rad. Oncol. 7 (2012)
146-166.
[2] a) G. W. Kalbalka, et al. J. Neuro-Oncol. 33 (1997)
153-161. b) P. Bendel, NMR in Biomed. 18 (2005) 74-82.
[3] Y. Sawamoto, master thesis, Tokyo University of
Science (2016)[4] T. Tanaka et al., Eur. J. Inorg. Chem. 1819-1834(2016).
[5] M. Kitamura et al., Inorg. Chem. 50 (2011)
11568-11580.
[6] T. Tanaka et al., Eur. J. Inorg. Chem. in press (2016)
Fig 1. Deboronation reaction of carborane derivatives 1

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CO6-6

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

T. Takahashi and N. Miyoshi1(2) Sample preparation: A cryo-sectioned (thickness=100m) tissue was prepared from the raw C6 glial tumor
model using a Cryo-section Maker (Leica) and wassealed sandwich-type with saran-wrap film (thickness=10m), under freezing condition (-20 C) before the measurements.

Research Reactor Institute, Kyoto University
1
Research Center for Development of Far-Infrared Region, University of Fukui

INTRODUCTION: The accelerator-based radiation
source in the millimeter and terahertz wave region hasvery attractive feature for the spectroscopy. Coherent
transition radiation (CTR), which has been emitted fromthe short bunches of electrons at the KURRI-LINAC, hasbeen used to observe the transmittance spectra of a sectioned tissue of raw brain tumor C6 model as a collaborate study in the research reactor institute, Kyoto university. The absorption spectra in the sub-terahertz region
had been not so clear for the raw tumor tissue although
Ashworth-PC. et al. [1] had reported for the excised human breast cancer by a terahertz pulsed spectroscopy
observed at 320 GHz, which was estimated a longer relaxation time component of the induced electricity forwater molecules [2-3] in the raw tumor tissue for three
years at the linear analysis.
We also estimated what kind of water molecules become
dominant in the viable and necrotic cancer regions by thedifferent measurement method as an aim of 2D mapping
study.
EXPERIMENTS: (1) Instrument of Near-field intera-hertz region: The experiment was performed at thecoherent radiation beamline [4] at the 40-MeV L-band
linac of the Research Reactor Institute, Kyoto University.
The width of the macro pulse and the repetition rate of
the electron beam were 47 ns and 60 Hz, respectively.
The charge of a bunch was 1.5 nC. The THz-wave source
was CTR emitted from an aluminum foil with 15-μm
thickness. The radiation was detected by a liquid helium
cooled Si bolometer. The conical cone with an aperture
260 μm in diameter was used as the illumination probe
and its F-number was 2.5. The spectrum of CTR was
measured by a Martin-Puplett type interferometer. The
schematic diagram of the experiment was shown in Fig.1.

RESULTS: The photograph of the cryo-sectioned tissue
with the measurement points was shown in Fig.2. The
spectra were measured at every point. The 2-dimensional
imaging of the observed intensity at the wavenumber of
19 cm-1 was shown in Fig.3. The comparison between the
spectral image and the H.&E.-stained Image is now progressing.

Fig.2 The photograph of the cryo-sectioned tissue withthe measurement points.

Fig.3 The 2-dimensional spectral imaging at the wavenumber of 19 cm-1.

REFERENCES:[1] Phillip C. Ashuworth, et. al., Optics Express, 17(14):12444-12454 (2009).
[2] Toshiko Fukasawa, et al., Phys. Rev. Let., 95: 197802(2005).
[3] Hiroyuki Yada, et al., Chem. Phys. Let., 464: 166-170(2008).
[4] T. Takahashi, et al., Rev. Sci. Instrum. 69 (1998)
3770.

Fig.1 The schematic diagram of the experiment.

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CO6-7

Protein Profiling and Drug Delivery on Cellular Apoptosis in Human Vitreous

T. Hisatomi, T. Tachibana, T. Ishibashi and
N Fujii2
Graduate School of Medical Sciencesm
Kyushu University,
1Graduate School of Science, Kyoto University
Research Reactor Institute, Kyoto University

INTRODUCTION: Protein profiling in human vitreous
may change in various vitreoretinal diseases. Regmatogenous retinal detachment is a severe retinal disease due tomassive loss of retinal photoreceptors. Photoreceptor loss
is mainly caused via molecular cascade to apoptosis.
During this process, many proteins are released or accumulated in vitreous. Since photoreceptor apoptosis isinitiated by activation of P2X7 receptor, P2X7 receptor
blocker, brilliant blue G (BBG) may have therapeutic
effects on retinal detachment. In this study, we examined
protein profiling and drug delivery in human vitreous
samples obtained during vitreous surgeries.

REFERENCES:
1)Notomi S, Hisatomi T, Kanemaru T, Takeda A, Ikeda
Y, Enaida H, Kroemer G, Ishibashi T.
Critical Involvement of Extracellular ATP Acting on
P2RX7 Purinergic Receptors in Photoreceptor Cell Death.
Am J Pathol. 2011 Dec;179(6):2798-809.
2)Notomi S, Hisatomi T, Murakami Y, Terasaki H, Sonoda S, Asato R, Takeda A, Ikeda Y, Enaida H, Sakamoto T, Ishibashi T.
Dynamic increase in extracellular ATP accelerates photoreceptor cell apoptosis via ligation of P2RX7 in subretinal hemorrhage. PLoS One. 2013;8(1):e53338.

EXPERIMENTS:
Human vitreous samples are examined by HPLC and theresults are compared between controls and various diseases. BBG concentration was analyzed with LC.

RESULTS:
The analysis showed major reported vitreous protein inhuman samples. The time-dependent changes of BBG are
also observed by LC. The BBG concentration in eye andblood was also compared in the dose-dependent manner.
Further studies are needed for detailed analysis in vitreoretinal diseases.

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