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Introduction
Recently, accumulating evidence has suggested that in
the diabetes state, oxidative stress increases in pancreatic
β-cells which have the lowest intrinsic antioxidant capacity
compared with other tissues[1,2].
At the same time, more and more large-scale studies have shown that angiotensin II
receptor blocker (ARB) evoke a significant risk reduction of
newly diagnosed diabetes when compared with β-blocker
calcium channel blocker or placebo, as demonstrated in the
LIFE (Losartan Intervention for Endpoint Reduction in
Hypertension) study, where losartan vs β-blocker, resulting
in a 25% reduction in the incidence of developing
diabetes[3]; the VALUE (Valsartan Antihypertensive Long-term Use
Evaluation) study, where valsartan vs amlodipine, resulting
in a 23% reduction of incidence[4]; and the CHARM
(Candesartan in Heart Failure — Assessment of Mortality
and Morbidity) trial, where candesartan vs placebo,
resulting in a 22% reduction of
incidence[5]. These trials have demonstrated that blockade of the renin-angiotensin
system (RAS) protects against the development of diabetes in
"at-risk" patients with hypertension, but the confirmative
mechanism of ARB in preventing diabetes remains equivocal;
it may lie on mitigation of islet fibrosis, oxidative stress and
β-cell apoptosis, or increase of islet blood flow.
There have been some studies which have proven the
existence of a local RAS in the
islets[6,7]. Several RAS components including AT1 and AT2 receptors, ACE, and
angiotensinogen were identified in mouse pancreatic islets.
Furthermore, the AT1 receptors were specifically localized to
the islet β-cells demonstrated by real-time RT-PCR,
Western blotting and double immunofluorescence
staining[8]. Short-term infusion of Ang II impairs first-phase insulin release,
possibly through changes in intra-islet blood flow; chronic
exposure to Ang II increases oxidative stress in
β-cells, activates fibrogenesis, and promotes apoptosis. Each of these
processes has also been implicated in the progressive loss
of β-cell function observed in type 2 diabetes.
The aim of the present study is to investigate whether
and how chronic ARB treatment can attenuate the
deleterious influence of hyperactive local RAS in the diabetes state
and reverse hyperglycemia using the well-characterized
model of non-insulin-dependent diabetes, the genetically
obese leptin receptor-deficient (db/db) mice. We also aimed
to explore whether these benefits are dose-dependent and
blood pressure-independent.
Materials and methods
Animals Forty-four male genetically diabetic, specific
pathogen-free female C57BL/KsJ-db/db mice weighing
35_40 g, and of 8 weeks of age, were purchased from CLEA
Japan Inc (Tokyo, Japan). These mice were randomized to
the ARB candesartan cilexetil 1 mg/kg, candesartan cilexetil
10 mg/kg, manidipine 10 mg/kg and placebo vehicle (5%
arabic gum) via gavage for 6 weeks. Eleven C57BL/KsJ-db/m
mice treated with placebo acted as nondiabetic controls. The
animals were housed in specific pathogen-free barrier
facilities at 23 °C with a light-dark cycle of 12:12 h and free access
to food and water. Ethics approval was granted by the
Animal Experimental Committee of the Medical Department of
Juntendo University (Tokyo, Japan). The animals were
handled according to the Principles of Laboratory Animal
Care (NIH Publication No 85_23, revised 1985).
Measurement of physiological and biochemical
parameters Body weight and random blood glucose were
measured every week. Systolic blood pressure (SBP) and heart
rate were measured at 2 h and 5 h after gavage at the starting
point and terminal, and the mean SBP was calculated.
Intraperitoneal glucose tolerance test (IPGTT)
After 6 weeks' treatment , an intraperitoneal glucose injection was
given after a 15 h overnight food deprivation at a dose of
0.5 g/kg body weight. Blood samples were taken at time 0
(before the glucose injection) and at 30, 60, and 120 min after
the glucose injection by tail cut. Glucose levels were
measured in whole blood with a compact glucose analyzer (One
Touch Ultra Glucose Meter, LifeScan Inc, Milpitas,
California, USA). Plasma insulin concentration were measured by a
commercial insulin ELISA kit (Morinaga ,Yokohama, Japan).
Immunohistochemistry Immunochemical detection of
insulin, p22phox protein,
gp91phox protein, 8-hydroxy-2'-deoxyguanosine (8-OHdG), 4-hydroxynonenal (4-HNE)
modified protein and CD31 were performed using the
avidin-biotin complex method. After the animals were deeply
anesthetized, the pancreases were removed and postfixed in
4% paraformaldehyde for 4_6 h and embedded in paraffin
and sectioned into 5 μm slices. Formalin-fixed tissue
sections were deparaffinized with xylene and rehydrated with
graded ethanols. Endogenous peroxidase activity was
blocked with 0.3% hydrogen peroxide; 10% normal goat
serum or 10% normal rabbit serum was applied for blocking
nonspecific binding for 10 min at room temperature. Then
the sections were incubated overnight for 14 h at 4 °C with
primary antibodies diluted in PBS containing 1% bovine
serum albumin (BSA). Primary antibodies included guinea pig
antihuman insulin antibody (1:2000, LINCO Inc, St Charles,
MO, USA), polyclonal goat anti-human
p22phox antibody, polyclonal goat anti-human
gp91phox antibody (1:100, Santa
Cruz Biotechnology, Santa Cruz, CA, USA), mouse
anti-human 8-OHdG monoclonal antibody (10 μg/mL; Japan
Institute for the Control of Aging, Fukuroi, Japan), mouse
anti-human 4-HNE monoclonal antibody (25 μg/mL, Japan
Institute for the Control of Aging, Japan), and purified rat anti
human CD31 antibody (1:200, BD Biosciences, Tokyo, Japan).
After washing with Phosphate Buffered Saline Tween
(PBST) 3 times, the sections were incubated with biotinylated
secondary antibody for 30 min at room temperature. Secondary
antibodies included biotinylated goat anti-guinea pig IgG
antibody (1:1000, Chemicon, Temecula, CA, USA),
biotinyl-ated rabbit anti-goat IgS antibody (1:200, Dako,Tokyo, Japan),
biotinylated goat anti-mouse IgS antibody (1:200, Dako,
Japan) and biotinylated goat anti-rat IgG
antibody (1:200, Cosmo Bio Co, Tokyo, Japan). Staining was completed
using 3,3'-diaminobenzidine (Sigma, USA) as the chromogen.
Sections were lightly counterstained with Mayer's
hematoxylin (Wako Co, Tokyo, Japan), dehydrated and mounted.
Some sections were used for Azan staining.
Morphometric analysis All sections were analyzed for
staining using light microscopy (E800, Nikon, Tokyo, Japan)
connected to an XYZ controller and a digital camera (Sony,
Tokyo, Japan). Digitized images were then captured as
digital images using Axiovision 4.3 software (Carl Zeiss Vision,
Hallbergmoos, Germany). At least 15 islets per mouse
pancreas and 3 mice per group were randomly chosen, and
therefore, at least 45 islets per group were analyzed.
Cells positive for 8-OHdG staining were quantified by
the presence of a dark brown nuclear stain. Observations
were made from a minimum of 45 islets, and when quantified,
were expressed as a percentage of the total number of islet
cells.
The intensity of immunoreactions of 4-HNE,
p22phox, p47phox and
gp91phox within the islets were graded
semiquan-titatively as follows: negative (score 0); weakly positive
(lightly stained, but clearly differentiated from negative
background, score 1); moderately positive (strongly stained
area=2 places, score 2); and strongly positive (strongly
stained area=3 places, score 3). Scores from a minimum of 45
islets were averaged for the evaluation of tissues from each
subject. The names of photoes for the morphometric
analysis were made single-blinded to the observers and
agreement between the two observers was greater than 90%.
The β-cell mass analysis was determined on an
insulin-stained section and was estimated by the following formula:
islet β-cell mass (mg)=the area of β-cells/the area of the whole
pancreatic area×pancreas weight (15 sections in each group).
The area of insulin-stained pancreatic islets was
automatically measured using image analysis software (Image-Pro
Plus 5.0.1, Planetron, Tokyo, Japan). Insulin staining
density was measured by image analysis software (Scion Image
B 4.0.3 for Windows, Scion Corp, Frederick, Maryland, USA)
with 30 islets in each group. CD31 staining intensity was
calculated by the following formula: CD31 staining intensity
(%)=the area of CD31 staining/the area of islet (30 islets in
each group).
Electron microscopy The pancreas were removed after
heart perfusion and fixed in a solution of 2.5% glutaraldehyde.
The pancreas were cut into pieces of approximately 1
mm3.The pieces were fixed in 2.5% glutaraldehyde buffered to pH
7.4 with phosphate buffer for 2 h, and treated with osmium
tetroxide for 2 h at 4 °C. The tissues were dehydrated with
graded concentration ethanol, and then embedded in Epok
812. Thin section were cut with a Leica Ultracut UCT (Leica
Mikrosysteme Gmbh, Wetzlar, Germany) with a diamond knife
and stained with uranyl acetate followed by lead citrate.
Electron micrographs were taken with a JEOL JEM-1200EX
Electron Microscope (JEOL, Tokyo, Japan) operated at 80 kV.
The relative volume of mitochondria was calculated by
counting the area of mitochondria on random micrographs at ×20
000 magnification using Image-Pro Plus software (4
micrographs were used in each group).
Statistical analysis All values are given as mean±SEM.
All statistical analyses were performed with SPSS for
Windows 11.0 statistical software package (Chicago, Illinois,
USA). One-way ANOVA was applied for multiple
comparisons of data, while Student's t-test was used for pair-wise
comparisons. For all comparisons, a P value of less than 0.05
was considered statistically significant.
Results
Physiological and biochemical parameter
At baseline, body weight, SBP, heart rate and blood glucose
concentrations were similar in the db/db control group, ARB 1 mg/kg
group, ARB 10 mg/kg group and manidipine group. After 6
weeks' treatment, there was no significant change in body
weight in all the groups, and the db/db control group showed
a significant SBP increase (P=0.002) ,while the extent of blood
pressure lowering was almost the same in the ARB 10 mg/kg
group and manidipine group (P=0.604). At the starting point
of 8 weeks' of age, the blood glucose concentration exceeded
26 mmol/L in all db/db mice, which meant that diabetes had
already reached a relatively severe state (Table 1).
Glycemia deteriorated continuously in the db/db control
mice during the study; it went above 30 mmol/L at the end of
second week and was almost 4.3 times as that of db/m
littermates of the same age at the terminate. Blood glucose
concentrations of the candesartan 1 mg/kg treated group began
to indicate slight amelioration after 2 week's treatment, and
the benefit appeared more legible at the terminal of study:
glucose concentration of the candesartan 1 mg/kg treated
group was significantly lower than that of the db/db control
mice (P<0.05) after 6 weeks' treatment (Figure 1). There was
no statistical significance observed in the candesartan 10
mg/kg and manidipine-treated groups when compared with
the db/db control group.
IPGTT The blood glucose concentration at 120 min after
glucose load was significantly reduced by candesartan 1
mg/kg treatment when compared with that of the db/db
control mice. Simultaneously-measured insulin concentration
did not decrease, but rather tended to increase, although the
difference was not significant. These results suggested that
the improvement of glucose tolerance by candesartan was
at least in part due to the improvement of β-cell function.
The candesartan 10 mg/kg and manidipine-treated group did
not show any statistically significant improvement on
glucose tolerance (P=0.062, Figure 2).
β-cell mass and insulin staining Diabetes is associated
with characteristic and progressive changes in the structure
of pancreatic islets. Such changes include loss of definition
of the islet boundary, and a decrease of β-cell mass.
β-cell mass was quite low in the db/db control group, and it was
rescued greatly after 6 weeks' treatment by both candesartan
1 mg/kg and 10 mg/kg (P<0.01 vs
db/db control, Figure 3). In the db/m mice, insulin staining was strong and symmetrical,
while it was sparse and asymmetric in the db/db mice treated
with placebo (Figure 4A). Treatment with candesartan also
notably improved staining density of insulin in the islets of
db/db when compared with placebo (P<0.01), although it
was still weaker than that of the db/m mice. Manidipine
treatment did not show any effect on either β-cell mass or
insulin intensity in the islets (Figure 4).
Immunohistochemistry of oxidative stress related
markers 8-OHdG is used as the most sensitive marker of
oxidative stress-related DNA injury. Positive staining was found
exclusively in the nuclei of β-cells (Figure 5A). The
percentage of dark brown nuclei in the db/db control group reached
48%, and when candesartan was used, the positive ratio
markedly declined to 20% and 12%, which was similar to that
of the db/m mice (14%). Manidipine treatment also provided
moderate relief of oxidative stress-related DNA injury (Figure
5B).
The 4-HNE modified protein is a marker of lipid
peroxida-tion products. According to the results of this study, the
positively stained area was dispersed among cytoplasm in
the islets of the db/db control group. Interestingly, the
4-HNE modified protein greatly diminished when treated with
either candesartan or manidipine (Figure 6).
Gp91phox and p22phox are membrane-binding subunits of
NADPH oxidase complex and they are thought to be
important for the NADPH oxidase to work. The positive staining
area appeared to be in the cytoplasm of the islet cells (Figures
7A, 8A). When semiquantitative analysis was taken into
account, the islets of the db/db control group exhibited
strongest gp91phox and
p22phox staining. Candesartan 1 mg/kg
treatment decreased the intensity of staining to almost a similar
extent of the db/m mice, and candesartan 10 mg/kg treatment
further ameliorated it. Manidipine treatment showed a less
notable, but significant reduction of expression of
gp91phox and p22phox (Figures 7, 8).
All these oxidative stress-related markers decreased with
candesartan treatment and this benefit was shown to be in a
dose-dependent manner.
Fibrosis Azan staining showed that in the islet of the
db/m mice, neither intra-islet nor around the islet was
blue-stained collagen found. In the db/db control mice,
blue-stained stripes were diffusely observed both intra-islet and
around the islet, which meant severe fibrosis. The
candesartan-treated db/db mice manifested obvious
alleviation of fibrosis, especially intra-islet. Manidipine treatment
hardly attenuated islet fibrosis (Figure 9).
Intra-islet blood supply Endothelial cell marker CD31
(PECAM-1) staining was used to evaluate density of blood
vessels within the islets. The islets of the db/m mice held
abundant blood vessels while the lack of blood supply was
discovered in the islets of the db/db mice. Candesartan
treatment restored the amount of vasculature to a relative high
level, similar to that of the db/m mice, while no improvement
was found with manidipine treatment (Figure 10).
Ultrastructure of pancreatic β-cells
Previous studies have also described ultrastructural changes within
β-cells that are associated with diabetes. These changes include
proliferation and hypertrophy of mitochondria, hypertrophy
of Golgi complexes, extensive endoplasmic reticulum and
severe reduction of the number of insulin secretory granules.
We observed similar derangements in the β-cells of the
db/db control mice. Aggregates of lysosomes were also observed,
a common feature of cells in the early stages of necrosis.
β-cells from the candesartan-treated animals exhibited improved
granulation and less remarkable endoplasmic reticulum and
Golgi bodies (Figures 11, 12A). Furthe-rmore, candesartan
treatment significantly relieved the swelling of mitochondria
(P<0.01) to nearly the same as that of the db/m group (Figure
12B). Not only did the volume decrease, but the percentage
of normal-shaped mitochondria greatly increased in the
candesartan-treated animals. The ultrastructure abnormity
of the manidipine treated β-cell was similar to that of
the db/db control.
Correlation analysis was performed and found that
mitochondria volume was strongly positively correlated to
oxidative stress markers including 8-OHdG
(r2=0.989, P<0.01; Figure 13),
p22phox (r2=0.925,
P <0.05) and gp91phox
(r2=0.943, P<0.05) staining intensity.
Discussion
We found that candesartan treatment effectively
protected pancreatic β-cell function failure independent of blood
pressure lowering, and revealed the potential mechanism of
the protective action of ARB by analyzing the status of
oxidative stress, fibrosis, blood supply and the ultrastructure
of the islets. The activity of the local RAS was an important
determinant of structure and function in a range of organs,
including the heart, kidneys and adrenals. Apart from its
potent vasoconstrictor actions, some studies revealed that
Ang II had several novel functions: stimulation and
inhibition of cell proliferation, induction of apoptosis, generation
of reactive oxygen species, regulation of hormone secretion,
and pro-inflammatory and pro-fibrogenic actions. Recently,
an intrinsic RAS was demonstrated in the pancreatic islets
which have the lowest intrinsic antioxidant capacity. Such a
local islet RAS, if activated, may drive islet fibrosis and
reduce islet blood flow, oxygen tension, and insulin
biosynthesis. Kampf et al demonstrated that endogenous
levels of Ang II exerted detrimental effects on islet blood
perfusion in transplanted mouse
islets[9]. Moreover, the
activation of an islet RAS may accelerate the synthesis of
reactive oxygen species, aggravate oxidative stress-induced
β-cell dysfunction and apoptosis, and thus contribute to the
islet failure seen in type 2
diabetes[10]. Accordingly, blockade of the RAS could contribute to the development of novel
therapeutic strategies in the prevention and treatment of
patients with diabetes.
Despite the appearance of severe glycemia, even at 8
weeks of age, candesartan successfully improved glucose
tolerance in diabetic db/db mice, although this protection
was not quite enough to reverse the state of diabetes to
normal. In any case, it assuredly provided some cooperative
evidence for the protection of ARB on diabetes observed in
large-scale epidemiological trials. We summarized a model
showing the proposed roles of Ang II in pathogenesis of
β-cell dysfunction and will discuss it in detail (Figure 14).
NADPH oxidase was originally discovered in neutrophils
where it plays a vital role in nonspecific host defense by
respiratory burst. In nonphagocytes under physiological
conditions, NADPH oxidase was maintained at low levels by
feedback of its production of reactive oxygen species (ROS),
whereas the feed-forward mechanism may work to generate
high levels of oxidative stress in a variety of diseases
including atherosclerosis and inflammation. A recent study
demonstrated that β-cells expressed
p22phox and gp91phox, the membranous components of the NADPH
oxidase[2]. In β-cells, glucose promotes
the production of ROS by protein kinase C (PKC)-dependent activation of NADPH oxidase. It
means that high glucose concentration in β-cells aggravates
the generation of ROS and leads to damage of β-cells. Like
glucose, Ang II also can increase the activity of NADPH
oxidase in islets by either PKC-dependent mode or direct
activation. The downstream effects of the activity of NADPH
oxidase include increased ROS generation and following
NF-kB and p38-MAPK activation, which lead to a vicious cycle
resulting in inflammation, insulin resistance and
β-cell dysfunction[11]. In our study, the existence of
gp91phox and p22phox were confirmed in
β-cells by immunohistochemical staining. NADPH oxidase expression was much stronger in
the db/db control mice than in the db/m mice, and candesartan
treatment reduced it significantly in a dose-dependent manner.
This evidence supports our view that candesartan decreases
the activity of NADPH oxidase by the inhibition of local
RAS in islets.
Other oxidative stress markers such as 8-OHdG and the
4-HNE modified protein are also used widely in the
evaluation of oxidative stress. In nuclear and mitochondrial DNA,
8-OHdG, an oxidized nucleoside of DNA, is the most
frequently detected and studied DNA lesion. A recent study
reported that elevated 8-OHdG correlated with
hypergly-cemia, and the severity of diabetic
nephropathy[12]. In present study, candesartan treatment was proven to be very
effective in reducing 8-OHdG levels which increased in the islets
of the db/db mice. The same results were observed in 4-HNE
staining. 4-HNE was proven to be a highly toxic lipid
peroxi-dation product and second toxic messenger of free radicals.
This multifunctional molecule, which derives from the most
represented class of polyunsaturated fatty acids in the
membranes, is potentially able to undergo a number of
reactions with proteins, phospholipids and nucleic acids, and
these modifications induce strong inhibitions of several
enzymatic activities, calcium overload and
apoptosis[13]. Thus, candesartan treatment comprehensively attenuated
oxidative stress and the harm caused by ROS and lipid
peroxidation. With RAS inhibition, the great amelioration of
oxidative stress resulted in a protective effect on
β-cells, which manifested as an increase of islet mass.
In the present study, we provide evidence that
cande-sartan improves angiogenesis in the islets of diabetic mice.
The reason for the lack of neovascularization in the db/db
mice may be that Ang II not only increases vasoconstrictive
factors such as endothelin-1, but also decreases vasodilative
and pro-angiogenic factors as PGI2 and bradykinin. Ang II
deprives bradykinin of postischemic neovascularization in
local islet at low oxygen tension state caused by
vasocons-triction. Therefore, candesartan rescued islet vasculature
by activating the local bradykinin pro-angiogenic pathway.
This opinion has also been proven in several studies where
skeletal muscle and hind limb ischemia were
observed[14,15]. The inhibition of local RAS in islets also brings about
mitigation of islet fibrosis. Ang II upregulates expression of
TGF-β and aggravates extracellular matrix accumulation
intra-islet and around the islet. Islet fibrosis process parallels
the duration of diabetes, and leads to severe β-cell
dysfunc-tion. Candesartan treatment effectively avoided severe islet
fibrosis by the inhibition of local RAS in the islets.
Mitochondria plays an important role in the development
of type 2 diabetes. We observed remarkable increased
mitochondria volume in the islets of db/db diabetic mice, which
was in agreement with early
studies[16_18]. It is speculated in an early study that
the enlargement of mitochondria in β-cells is induced by demands to synthesize and secrete insulin,
conformed to proliferative Golgi apparatus and rough
endoplasmic reticulum, and depletion of secretory
granules, but a recent theory seems to be more credible: mitochondria is one
of the main sources of reactive oxygen species. It has been
estimated that up to 2% of the total mitochondrial oxygen
consumption results in ROS
generation[19]. Mitochondria are also the cellular component most extensively affected by
increased concentrations of ROS. The inner mitochondrial
membrane is believed to be particularly susceptible to
oxidative damage even under physiological conditions, for it is a
major site where ROS, and a high content of
polyunsaturated fatty acids, accumulate and react with each other, thus
yielding a large amount of stronger lipid peroxidation
products which incur alterations in the structure integrity of
mitochondrial membranes, causing irreversible swelling and
disruption. A previous study demonstrated that oxidative
stress induced in vitro by
ADP-Fe2+ in mitochondria strongly initiated a significant increase of lipid peroxidation (measured
by O2 consumption and thiobarbituric acid reactive species
formation) and concurrent increase of mitochondria
volume[20]. Another study proves that antioxidative agents such as
Ginkgo biloba extract (EGb 761) succeeds in protecting
mitochondria in cells of liver and heart from enlarging using
diabetic model animals[20,21]. Ang II increases NADPH
oxidase activity and decreases NO levels, and both lead to
aggravation of oxidative stress. Furthermore, the decline of
NO levels directly upregulates the activity of mitochondria
by removing the inhibition of cytochrome oxidase and
aconitase in the TCA cycle, thus resulting in more ROS
production and severe mitochondria damage. We described for
the first time that the AT1-receptor blocker candesartan
effectively prevented the mitochondria of β-cells from
swelling and disruption in a dose-dependent manner, and
maintained almost the normal size as those of the db/m mice.
Moreover, the percentage of normal-shaped mitochondria
also increased when compared with the untreated mice. The
mechanism may lie on direct relief of mitochondrial
hyper-action and reduction of the activity of NADPH oxidase and
their downstream ROS products such as 4-HNE.
The discrepancy of effects on attenuating oxidative stress
and on glucose tolerance as well as β-cell mass may be due
to the overdose of candesartan when 10 mg/kg was used.
Candesartan 10 mg/kg is hardly clinically achievable and
may cause no harm to β-cell function. Results of CD31
staining demonstrated a decreased islet vasculature when
candesartan dosage was added to 10 mg/kg. Also, some
studies reported that candesartan could reduce vascular
endothelial growth factor (VEGF) expression and subsequent
angiogenesis in different
organs[14,22,23], and this inhibition may partly counteract endothelium protection benefited from
the alleviation of oxidative stress. Yet more studies are
needed to reveal the accurate effects of candesartan on islets.
In conclusion, after diabetes was initiated, chronic
candesartan treatment could not reverse the state of diabetes,
which is consistent with several studies using other kinds of
ARB[24,25], but 6 weeks' candesartan treatment effectively
improved glucose tolerance, remarkably decreased oxidative
stress in β-cells, and prevented damage from the excess of
NADPH oxidase and lipid peroxidation products, thus
leading to the preservation of islet mass; we first found that
ARB effectively prevented ultrastructure damage in
β-cells of diabetes. All these benefits appear to be independent of
blood pressure reduction, and the antioxidation effect seemed
to be in a dose-dependent manner. The characteristics of
candesartan may make itself a novel therapeutic means for
protecting against progressive β-cell failure in diabetes.
Acknowledgements
We are grateful to Mrs Naoko DAIMARU, Yukiko TOYOFUKU and Eriko MAGOSHI for their skillful technical
assistance.
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