 1 Type II
Receptor Gene in Alveolar Lining Epithelial Cells of Idiopathic
Pulmonary Fibrosis
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Abstract |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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It has been reported that transforming growth factor (TGF)-
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which plays an integral role in the pathogenesis of idiopathic pulmonary fibrosis (IPF), suppresses proliferation of alveolar epithelial cells in vitro. Although hyperplastic lesions of alveolar lining epithelial cells (ALECs) are characteristic pathologic features of IPF, the mechanism of their involvement in the
pathogenesis has not yet been extensively studied. On the assumption that the hyperplastic ALECs have escaped from the
growth-inhibitory effects of TGF-, we searched for mutations
in the microsatellite of the TGF- receptor type II (TRII)
gene. To detect a deletion in the polyadenine tract in exon 3 of the TRII gene, cells were isolated by microdissection from
lung sections of IPF patients, and DNA was extracted from
these cells and amplified by high-fidelity polymerase chain reaction. A total of 121 sites of hyperplastic ALECs from 11 IPF
patients were analyzed, and a one-base-pair deletion was detected in nine sites from five patients. The mutation was also
detected in smooth muscle-like cells of the thickened pulmonary artery. In some tissue areas where the deletion was detected, low TRII expression was confirmed by immunohistochemical staining. These data suggest that microsatellite
instability in the TRII gene occurred in some lesions of hyperplastic ALECs in IPF, although at a low incidence, and that this
genetic disorder might play a partial role in the pathologic
changes of IPF.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Idiopathic pulmonary fibrosis (IPF) is a progressive disease of the lung interstitium of unknown etiology with nonspecific and diverse pathologic features (1, 2). It has been
reported that IPF is associated with changes in various
kinds of cytokines and growth factors (3). Transforming
growth factor (TGF)- is one of the critical factors that
precipitate the process of lung fibrosis of IPF. Several investigators have reported that production of TGF- is elevated in pulmonary epithelial cells and alveolar macrophages in the lungs of IPF patients (4). TGF- is known
to play an integral role in fibrosis of various tissues
through its effects on fibroblast proliferation and promotion of collagen synthesis (7). Previously we reported that
intratracheal instillation of plasmid complementary DNA
coding for the TGF-1 gene into the rat lungs caused apparently fibrotic changes in the interstitium (8).
Although hyperplastic lesions of alveolar lining epithelial cells (ALECs) are characteristic and important pathologic features observed in lesions of IPF (1, 9), the biologic
significance of these lesions has not been fully elucidated. As
described earlier, TGF- is a critical factor for the pathogenesis of lung fibrosis, but its effects on ALECs are still
under investigation. A number of investigators have reported that TGF- exerts a growth-inhibitory effect on
various cells, including lung epithelial cells (10). These
observations suggest that TGF- is critical in the regulation of alveolar epithelial cell growth. Hence, the hypothesis that the alveolar epithelial cells that lose responsiveness
to TGF- can acquire a growth advantage and become hyperplastic has been advanced.
As one mechanism of deregulation of TGF-, mutations of the TGF- receptor type II (TRII) gene were
first reported in colon cancer (hereditary nonpolyposis colon carcinoma) (14, 15), and subsequently in various other
tumors (16). A deletion of one or more base pairs (bp)
in the polyadenine (A10) microsatellite tract in exon 3 is
the most frequent type of mutation. Further, microsatellite
instability of the TRII gene was found not only in neoplasms but also in a nonmalignant disease. McCaffrey and colleagues demonstrated that microsatellite instability of
the TRII gene is observed in lesions of atherosclerosis
and especially in restenotic vascular lesions (19). TGF- is
highly expressed in these lesions (20, 21). Moreover, it exerts a potent growth-inhibitory effect on smooth muscle-
like cells of the arterial wall in vitro (10). McCaffrey and associates suggested that the genomic instability might cause
a growth advantage of smooth muscle-like cells by deregulation of TGF-, and might play a critical role in the pathogenesis of this vascular disease (19).
In the pathogenesis of IPF, it is important and also of
great interest to examine whether mutation in the TRII
gene is observed in the hyperplastic ALECs, and whether
any such mutations are relevant to responsiveness to TGF-.
In the present investigation, to clarify the pathogenesis of
IPF, we analyzed the changes of TRII expression caused
by the microsatellite instability of the TRII gene in tissues obtained from the lungs of patients with IPF.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Study Population
Patients were diagnosed with IPF on the basis of clinical findings, diagnostic imaging, and histologic examinations. Lung specimens of 11 IPF patients, seven men and four women ranging in age from 44 to 79 yr (mean 62.3), were obtained by video-assisted thoracoscopic lung biopsy or open lung biopsy and diagnosed histologically as usual interstitial pneumonitis (1) at Osaka University Hospital, National Kinki-chuo Hospital, and National Toneyama Hospital from 1991 to 1998 (Table 1). As controls, surgically resected lung specimens from three patients with lung cancer were also examined. The protocol was approved by the ethics committees of the respective hospitals.
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Microdissection
The formaldehyde-fixed, paraffin-embedded lung tissues were cut into 1- or 3-µm-thick sections, and the sections were deparaffinized and visualized by hematoxylin and eosin (H&E) staining. Approximately 10 to 50 cells were microdissected under a microscope (Olympus, Tokyo, Japan) with a capillary glass tube using an electric micromanipulator (Narishige, Tokyo, Japan) from hyperplastic ALECs, bronchial epithelial cells, fibroblasts in fibrotic foci, and smooth muscle-like cells in thickened pulmonary artery (PA) wall by morphologic judgment (22). Bronchial epithelial cells and normal pulmonary artery wall cells from outside of the areas of tumor invasion in lung specimens of the patients with lung cancer were also obtained for controls.
Polymerase Chain Reaction Primers
Two pairs of primers were designed to cover the polyadenine microsatellite tract in exon 3 of the TRII gene. The sequence of
exon 3 of the TRII gene was reported by Lin and coworkers (GenBank No. M85079) (23). All oligonucleotides used as polymerase chain reaction (PCR) primers are listed later. Primer 3R
was rhodamine-labeled primer 3 (purchased from Takara, Kyoto, Japan).
Primer 1: 5'-CAGTTTGCCATGACCCCAAG-3';
Primer 2: 5'-CTTCTGAGAAGATGATGTTGTCAT-3';
Primer 3: 5'-CCCCTACCATGACTTTATTCTGGA-3';
Primer 3R: 5'-Rhodamine-CCCCTACCATGACTTTATTCTGGA-3';
Primer 4: 5'-CATTGCACTCATCAGAGCTACAGG-3'.
Standard DNA
For genomic standards, we used DNA extracted from normal human peripheral blood cells of a healthy volunteer, and a human colon carcinoma cell line, HCT116, which was obtained from
American Type Culture Collection (ATCC) (Rockville, MD;
ATCC number CCL-227). It has been reported that HCT116
cells have a 1-bp deletion in the polyadenine microsatellite tract
in exon 3 of the TRII gene (24). Buffer without DNA was also
amplified as a negative control.
Detection of the Deletion in the Microsatellite Tract by PCR
We performed high-fidelity PCR and restriction analysis (19). To
extract genomic DNA, cells obtained by microdissection were incubated with 10 µl of proteinase K (200 µg/ml) solution in 20 mM
Tris-HCl (pH 7.5), 1 mM ethylenediaminetetraacetic acid, and
0.5% Tween 20 at 37°C for 24 to 48 h. The mixture was then heated at 98°C for 8 min to inactivate proteinase K. For amplification of exon 3 of the TRII gene, nested PCR was performed.
The first PCR was performed in a 25-µl volume containing 10 µl
of extracted DNA solution, 1× PCR buffer, 0.2 mM of each deoxynucleotide triphosphate (dNTPs), 0.3 µM primer 1, 0.3 µM
primer 2, and 5 U of KOD polymerase (Toyobo, Tokyo, Japan).
The second PCR was also performed in a 25-µl volume containing 1 µl of the first PCR solution, 1× PCR buffer, 0.2 mM of each
dNTP, 0.5 µM primer 3R, 0.5 µM primer 4, and 5 U of KOD polymerase.
The amplification program consisted of 30 cycles of 94°C for
1 min, 61°C for 1 min, and 72°C for 20 s, performed using a Thermal Cycler (Perkin-Elmer, Norwalk, CT). For control template, we
used 180 pg of standard DNA extracted from normal human peripheral blood cells and HCT116 cells. The PCR products were
digested with 2.4 U of AluI (Nippongene, Tokyo, Japan) at 37°C
for more than 3 h. After denaturation at 94°C for 4 min, 2-µl aliquots of the PCR products were applied to 6% acrylamide/8 M
urea sequencing gels, and electrophoresed at 2,500 V for 2.4 h.
The densities of the bands were quantified by measuring the absorbance at 605 nm with the FMBIO II Multi-View system
(Takara). The actual value was corrected relative to values of
bands generated from standard DNA (normal and HCT116) in
every electrophoresis. In this method, the length of the PCR
product from the normal TRII gene after AluI digestion is 99 bp,
and that from the TRII gene with a 1-bp deletion in exon 3 is 98 bp.
The deletion rate was expressed as the percentage of the density
of the mutant band (A9-98 bp) to the total of the density of the
wild-type band (A10-99 bp) plus the mutant band (A9-98 bp).
Using this method, non-negligible ladder bands besides the main band appeared after electrophoresis. In the cases of standard DNA, less than 8% of the counts of the primary band (normal 2.0 ± 1.9%, HCT116 1.4 ± 1.8%, means ± standard deviation [SD]) were detected in the 1-bp-shorter band, and approximately 20 to 30% of the counts (normal 27.6 ± 32.6%, HCT116 20.3 ± 34.4%, means ± SD) were detected in the 1-bp-longer band. Therefore, the deletion rates were calculated after the counts of the bands from samples were corrected by comparison with the control from standard DNA in every electrophoresis. Moreover, in samples of morphologically normal bronchial epithelial cells, the deletion rates after correction were less than 10.7% (2.3 ± 3.1%, mean ± SD). Therefore, samples with a deletion rate of over 20% were defined as deletion-positive. We detected no bands from samples of buffer without template DNA.
Immunohistochemical Staining
We performed immunohistochemical staining with antibody against
human TRII (C-16) (Santa Cruz Biotechnology, Heidelberg, Germany) and antibody against human TGF- (MAB240; R&D Systems, Minneapolis, MN), using VECTASTAIN Universal Quick
Kit (Vector Laboratories, Burlingame, CA) according to the manufacturer's protocol. This antibody against TRII recognizes the
C-terminal 16 amino acids of the intracellular domain. Briefly, sections of lung tissue of IPF were deparaffinized and hydrated through xylene and a graded alcohol series. After blockage of endogenous peroxidase with methanol containing 0.3% hydrogen peroxide, the sections were incubated for about 10 min in phosphate-buffered saline (PBS)-diluted blocking horse serum. Excess serum was discarded, and the sections were incubated overnight at 4°C with antibody against TRII diluted 1:200, antibody against TGF- diluted
1:100 in PBS, or PBS alone as a negative control. The sections were washed in PBS, and incubated in PBS-diluted biotinylated universal secondary antibody for 10 min. The sections were incubated in 3,3'-diaminobenzidine (Sigma, St. Louis, MO) solution as the substrate of the peroxidase until staining intensity developed, and then they were stained with hematoxylin and included.
DNA Sequencing
DNA sequencing was performed on three samples of the PCR products that showed deletion rates of almost 100%. A second PCR was performed under the same conditions as the first except for the use of primer 3 instead of primer 3R. The PCR products were separated by electrophoresis on low-melting-point agarose gels and extracted from the gels. The purified products were labeled with primer 3 or primer 4 by fluorescent dye-terminator chemistry using the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA), and electrophoresis was performed using the ABI 373 DNA Sequencing System (Applied Biosystems) according to the standard protocol.
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Results |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Deletion in TRII Gene in IPF
Clusters of hyperplastic ALECs in 138 sites from the 11 IPF patients were microdissected from the paraffin-embedded tissue sections, and the DNA templates extracted therefrom were amplified by nested PCR. We detected no bands after electrophoresis in samples obtained from 17 sites, so we examined whether deletions were present in the remaining 121 sites.
We detected the deletion mutations in the TRII gene
in nine samples from five IPF patients that showed deletion rates of 20 to 100% (Table 2). Representative results
of the analysis conducted are shown in Figure 1. Tissue
sections of the fourth site of case 3 (site 3-4) before and after microdissection are shown in Figure 1A. The results of
deletion analysis of sample 3-4 are shown in Figure 1B
(lane 3); this analysis revealed that the deletion rate was
65%. DNA samples obtained from blood cells from a
healthy volunteer (Figure 1B, lane 1, N) and a colon carcinoma cell line, HCT116 (Figure 1B, lane 2, H), were used
as controls. In the other sites of the hyperplastic ALECs of
case 3, for example, in representative samples 3-5 and 3-6 (Figure 1B, lanes 4 and 5, respectively), the deletion rates
were nearly 0%. Similar results from other case of ALECs
are shown in Figure 1C. The deletion rate in sample 1-18 was 27% (Figure 1C, lane 3), whereas the rates in the
other sites of the ALECs of samples 1-19 and 1-20 (Figure 1C, lanes 4 and 5, respectively), were 0%. In case 3, a
higher incidence of deletion mutations in ALECs was observed than in the other cases (Table 3).
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We could detect the bands in a total of 40 samples of smooth muscle-like cells in thickened PA wall out of 48 sites microdissected, and seven samples from three IPF patients showed deletion rates of 20 to 100% (Table 2). The proliferating cells in the PA wall had primarily smooth muscle-like characteristics. The analysis of a representative positive site of smooth muscle-like cells in the thickened PA wall (site 7-9) is shown in Figures 1D and 1E. By the same method described earlier, we detected a deletion rate of 24% in sample 7-9 (Figure 1E, lane 5). However, we could not detect any mutation in the other samples of ALECs or smooth muscle-like cells in the PA wall of case 7.
We also tested DNA samples obtained from 18 sites each of fibrotic foci or bronchial epithelial cells without morphologic abnormality, and we could not detect any mutation in any of the sites tested (Table 2).
Immunohistochemical Analysis of TRII Expression
We next tested TRII (cases 3, 5, and 6) and TGF- (cases
5 and 6) expression in ALECs. Most hyperplastic ALECs,
proliferating fibroblasts, and myofibroblasts in the area of
fibrotic change were strongly stained by immunohistochemical staining with antibody against human TRII. However,
in some lesions of ALECs there was locally decreased
staining intensity. We examined the level of TRII expression in a series of tissue slices by immunohistochemistry in
five out of nine sites of hyperplastic ALECs in which the
mutation in the TRII gene was present. In two of the five
sites we detected low expression of TRII protein.
A representative lesion of bronchiolization of ALECs
(site 5-7) and the section after microdissection are shown
in Figures 2A and 2B. Immunohistochemical staining was
performed with antibody against human TRII in the section adjacent to that shown in Figure 2B. At the site of the
bronchiolization, the staining intensity was significantly
weak (the center of Figure 2C; arrow). The analysis of
sample 5-7 showed a deletion rate of almost 100%. (Figure
2E, lane 5, arrow).
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A similar result was obtained at another site (site 6-3)
shown in Figure 3. The staining intensity with antibody
against human TRII was locally weak in the center of the
tissue section (Figure 3C, arrow). The deletion rate in
sample 6-3 was 20% (Figure 3E, lane 5, arrow).
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Additionally, we examined the extent of TGF- expression in a series of tissue slices by immunohistochemistry in
two sites of hyperplastic ALECs in which low expression of
the TRII protein was confirmed. Most of the bronchial
epithelial cells, many ALECs, and fibroblasts were positive
as previously reported (4). The level of expression of TGF-
protein at the ALECs with deletion in the TRII gene in a
series of slice was not necessarily higher than that in the
surrounding lesions of ALECs (Figures 2D and 3D).
DNA Sequencing
We performed DNA sequencing of the PCR products. In
the three samples in which the deletion rate was almost
100% (samples 3-30, 4-1, and 5-7), we determined the DNA
sequence of the portion bounded by the primers in exon 3 of the TRII gene. We detected a 1-bp deletion in the
polyadenine microsatellite tract (A9), which was previously reported in various types of cancer cells. A representative sequencing result in a case that had a deletion rate
of 100% (sample 5-7, the same sample shown in Figure
3E) is shown in Figure 4A.
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Discussion |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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We detected a deletion mutation of the TRII gene in the
hyperplastic ALECs and smooth muscle-like cells of the
thickened PA wall in IPF patients. The incidence of genomic instability we detected in ALECs was not so high
(nine of 121 sites). However, in seven out of 11 IPF patients, we detected more than one lesional site containing
the deletion mutation in either hyperplastic ALECs or
thickened PA.
To examine the microsatellite instability in the TRII
gene in microlesions of the tissue, we used a modification
of the method described by McCaffrey and colleagues (19).
In this investigation, the accuracy of our assay depended
upon amplification in the PCR step with a small amount of
template DNA obtained from paraffin-embedded tissues.
We could isolate no more than 100 cells because the hyperplastic lesions in IPF were relatively small. Additionally,
the lung tissues that we used were embedded in paraffin
and had been kept at room temperature. Hence, genomic
DNA in the tissue was likely to have been damaged and
degraded into fragments several hundred base pairs in
length (25). Inasmuch as we could not obtain longer fragments, we searched for deletion mutations only in exon 3 of the TRII gene.
We performed nested PCR with KOD polymerase to achieve amplification from a small amount of DNA. Because of 3'-5' exonuclease activity, this polymerase has 12-fold higher fidelity than Taq polymerase, and thus has fidelity almost equal to that of Pfu polymerase (according to the products' guides). The 3' end of rhodamine-labeled PCR products is often unstable after PCR, so the products were cleaved at the AluI site between the polyadenine tract and the 3' end. Despite our efforts to increase the accuracy of PCR amplification, the ladder of bands remained after electrophoresis. Shorter false-positive bands interfere with determining the presence or absence of a deletion more than longer bands. Fortunately, the amount of the shorter false-positive bands was less than 8% of that of the major bands in the cases of standard DNA, so this assay was suitable for detecting deletion-positive rates of over 20%.
The hyperplastic lesions of ALECs are characteristic
pathologic features of IPF (1, 9). Previous observations have
shown that TGF- is an important growth factor in the
pathogenesis of IPF (4, 8). It was reported that overexpression of TGF- occurs in hyperplastic ALECs (4, 5).
However, the proliferation of lung epithelial cells is negatively regulated by TGF-1 in vitro (10). To explain the
mechanism hyperplasia in ALECs, we assumed that the
hyperplastic ALECs escape from the growth-inhibitory effect of TGF-. There are two distinct types of TGF- cell-surface receptor, types I and II. Both receptors have serine/
threonine kinase activity and play an essential role in signal transduction of TGF- (26). A deletion mutation in
the TRII gene is one of the mechanisms responsible for
deregulation of TGF-.
A mutation in the TRII gene was first reported in colon cancer cells by Markowitz and associates (14) and Parsons and coworkers (15), and thereafter in various kinds of
cancers, such as gastric and pancreatic cancers (16).
Most mutations in the TRII gene were deletion of one or
more base pairs in the polyadenine microsatellite tract
(A10), and other mutations included microsatellite instability in (GT)3 repeats or point mutations. The deletion of
one or two base pairs in the polyadenine tract of the
TRII gene gives rise to a stop codon before the transmembrane domain. It was reported that cell lines with a
1-bp deletion in the polyadenine tract of the TRII gene,
such as HCT116, acquired resistance to TGF- (24, 30),
and it was also reported that overexpression of the dominant-negative TRII gene in various cells caused reduced TGF- responses, including those in a mink-lung epithelial
cell line, Mv1Lu (31). Together, these observations suggest that the deletion mutation in exon 3 of the TRII gene
is likely to cause hyporesponsiveness to TGF- and a cellular growth advantage. This type of deletion mutation in
the TRII gene was also detected in a nonmalignant disease, atherosclerosis, by McCaffrey and coworkers (19).
They proposed that microsatellite instability in the TRII gene disables growth inhibitory pathways, allowing monoclonal selection of a disease-prone cell type within some
vascular lesions.
Our present investigation demonstrated that the deletion
mutation in exon 3 of the TRII gene was also detected in
IPF. We also confirmed that TRII protein was expressed
weakly in some ALEC hyperplastic lesions which contained
the deletion mutation in the TRII gene. The signal transduction pathway from TGF- via TRII may possibly be
reduced or lost in these gene mutation-containing epithelial
cells, and the cells are thus predicted to acquire resistance to
TGF- and consequently a growth advantage in the presence of TGF- compared with other cells without such mutations. The majority of alveolar epithelial cells regenerated
as a consequence of lung inflammation. However, our observations suggest that a portion of these cells escaped from
the growth inhibitory effects of TGF- via acquired genomic instability of the TRII gene, resulting in overgrowth
and hyperplasia in some of these lesions.
We also detected the deletion in the TRII gene in the
thickened PA wall. There are a few reports about PA wall
thickening in IPF (9); however, the mutation in the TRII
gene in the coronary artery has been reported (19). It is
also possible that the growth advantage of smooth muscle-
like cells with TRII mutations is due to deregulation of
TGF- in some lesions of thickened PA wall. In fibroblasts
of fibrotic foci and bronchial epithelial cells, the mutation
was not detected. However, the number of specimens examined in this investigation may not have been sufficient
to reach a solid conclusion.
It is still unclear whether the deletion mutation in the
TRII gene and deregulation of TGF- in alveolar epithelial cells directly induces lung fibrosis. The number of lesions in which the ALECs had the deletion in the TRII
gene and deregulation of TGF- was limited. Therefore, it
is difficult to conclude that the deletion in the TRII gene
is directly responsible for the pathogenesis of fibrosis.
However, a few investigators have reported a relationship
between TGF- signal blocking and tissue fibrosis in animal models. Amendt and colleagues reported that a high
level of proliferation in the epidermis was correlated with
a very early onset of carcinoma development in keratin 5 promoter-controlled, epidermis-specific, dominant-negative TRII transgenic mice, and expression of TGF- protein was present in the majority of early carcinoma cells
(34). Bottinger and associates reported that in pancreatic
acinar cell-specific, dominant-negative TRII transgenic mice, expression of TGF-1 messenger RNA and protein
was markedly increased in acinar cells (35). Additionally,
pancreatic interstitial fibrosis was observed in the transgenic mice. These data raise the possibility that hyporesponsiveness to TGF- in epithelial cells may induce augmented expression of TGF- and consequently promote
interstitial fibrosis. On the other hand, in the present investigation we could not clearly confirm much higher expression of TGF- protein on the ALECs with the deletion in the TRII gene compared with those without the
deletion because the hyperplastic ALECs primarily expressed high levels of TGF- in IPF patients (4, 5). Therefore, we could not lead to the specific speculations of the
level of TGF- protein by immunohistochemical analysis. The fibrotic mechanism in IPF would have been much more
complicated compared with that in pure animal models due
to the involvement of various factors.
In conclusion, our observations suggest that a specific
genetic disorder and functional loss of TRII caused by
microsatellite instability may play a critical role in some lesions of ALEC hyperplasia in IPF patients. There have
been few reports of genetic disorders in IPF, but it is possible that genetic mutations of various factors, including the
TRII gene, concerned with cell growth and fibrosis occur
in IPF. IPF causes irreversible deterioration, and its mechanism is not fully understood. The results of the current
study offer a partial explanation for the pathophysiology
of the irreversibility of IPF.
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Footnotes |
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Address correspondence to: Masahide Mori, M.D., Dept. of Molecular Medicine, Osaka University Graduate School of Medicine (C4), Suita, Osaka, 565-0871, Japan. E-mail: morimasa{at}imed3.med.osaka-u.ac.jp
(Received in original form April 10, 2000 and in revised form September 8, 2000).
Abbreviations: alveolar lining epithelial cell, ALEC; base pair(s), bp; hematoxylin and eosin, H&E; idiopathic pulmonary fibrosis, IPF; pulmonary artery, PA; phosphate-buffered saline, PBS; polymerase chain reaction, PCR; standard deviation, SD; TGF- receptor type II, TRII; transforming growth factor, TGF.
Acknowledgments: The authors are grateful to Mr. T. Teramoto, Mr. M. Naka (National Kinki-chuo Hospital), and Mr. T. Hashimoto (National Toneyama Hospital) for their technical support. The authors also thank Miss Y. Habe for her secretarial work. This study was supported by a grant-in-aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan.
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References |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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