Human Molecular Genetics, 2000, Vol. 9, No. 20 3047-3054
© 2000 Oxford University Press
Caveolin-3 deficiency causes muscle degeneration in mice
National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo 187-8502, Japan and 1Department of Anatomy II, National Defence Medical College, Tokorozawa, Saitama 359-8513, Japan
Received 18 August 2000; Revised and Accepted 13 October 2000.
DDBJ/EMBL/GenBank accession no. AY007215.
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ABSTRACT |
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Caveolin-3 is a muscle-specific protein integrated in the caveolae, which are small invaginations of the plasma membrane. Mutations of the caveolin-3 gene, localized at 3p25, have been reported to be involved in the pathogenesis of limb-girdle muscular dystrophy (LGMD1C or caveolinopathy) with mild clinical symptoms, inherited through an autosomal dominant form of genetic transmission. To elucidate the pathogenetic mechanism, we developed caveolin-3-deficient mice for use as animal models of caveolinopathy. Caveolin-3 mRNA and its protein were absent in homozygous mutant mice. In heterozygous mutant mice, both the mRNA and its protein were normal in size, but their amounts were reduced by about half. The density of caveolae in skeletal muscle plasma membrane was roughly proportional to the amount of caveolin-3. In homozygous mutant mice, muscle degeneration was recognized in soleus muscle at 8 weeks of age and in the diaphragm from 8 to 30 weeks, although there was no difference in growth and movement between wild-type and mutant mice. No apparent muscle degeneration was observed in heterozygous mutant mice, indicating that pathological changes caused by caveolin-3 gene disruption were inherited through the recessive form of genetic transmission.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Caveolin-3 (or M-caveolin) is a muscle-specific protein of the caveolin family, which includes caveolins-1 and -2 (1–4). Caveolins-1 and -2 are abundantly expressed in adipocytes, endothelial cells and fibroblast cells (5). Caveolins-3 and -1 are closely related based on primary sequence homology, showing 65% identity and 85% similarity (2). Caveolin is the principal component of caveolae, which are 50–100 nm vesicular invaginations of the plasma membrane (4,6). Caveolin-induced caveolar formation appears to be isoform specific. Although recombinant expression of caveolin-1 or -3 in insect cells that lack caveolae is sufficient to induce caveolar formation, recombinant expression of caveolin-2 under the same conditions failed to induce the formation (7). During the process of caveolar formation, caveolin undergoes two stages of self-association or oligomerization (8,9). Shortly after caveolin synthesis, caveolin oligomerizes in the endoplasmic reticulum to form homo-oligomers of 300–350 kDa, each containing
14–16 individual caveolin monomers. At later stages, caveolin homo-oligomers interact with each other to form clusters. A 33 amino acid hydrophobic domain of caveolin-1, which spans the membrane, is thought to form a hairpin loop within the cell membrane, allowing both N- and C-terminal domains to face the cytoplasm (4). The N-terminal domain directs the formation of caveolin homo-oligomers (9), whereas the C-terminal domain acts as a bridge to allow these homo-oligomers to interact with each other, thereby forming a caveolin-rich scaffold (10). Through a common caveolin domain, termed the caveolin-scaffolding domain, caveolin binds and regulates several caveolin-associated signaling molecules (11,12). Caveolin-3 also shares the same characteristics. It is noteworthy that in addition to its direct binding to neuronal nitric oxide synthase (nNOS) (13), caveolin-3 indirectly binds with dystrophin (3,14,15), which is a product of the Duchenne muscular dystrophy gene (16), and also with the sarcoglycan complex, which is composed of four subunits and the loss of which causes muscular dystrophy (17). Recently, a study on human patients by Minetti et al. (18) revealed that mutations of the caveolin-3 gene, localized at 3p25, caused mild muscular dystrophy (LGMD1C or caveolinopathy) to be inherited in an autosomal dominant manner.
To better understand the function(s) of caveolin-3 and its role in the pathogenetic mechanism of caveolinopathy, we generated caveolin-3-deficient mice using a targeting technique. We found that the density of caveolae in skeletal muscle plasma membrane decreased as the amount of caveolin-3 decreased. Pathological changes were, albeit mild, clearly evoked in some selected muscles of homozygous mutant (cav3–/–) mice. However, overt pathological changes were not observed in muscles of heterozygous mutant (cav3+/–) mice. Therefore, we consider that pathological changes in our mice caused by caveolin-3 gene disruption are inherited in a recessive manner.
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RESULTS |
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Generation of cav3–/– mice
To design a targeting vector for the generation of cav3–/– mice, we characterized a P1 clone containing the murine caveolin-3 gene (cav3). Murine and human caveolin-3 have highly homologous amino acid sequences [95% identity (18) and 96% similarity (14)] and the structural organization of the gene consisting of two exons is shared by both species (14,19).
As shown in Figure 1A, we designed a targeting vector to disrupt exon 2, which encodes the caveolin-scaffolding and membrane-spanning domains and the C-terminal region and in which genetic mutations in LGMD1C patients have been identified (18). The targeting vector was introduced into the embryonic stem cell line and three independent clones were identified as homologous recombinants. cav3+/– mice with the germline transmission were interbred to obtain cav3–/– mice and the disruption of cav3 in the offspring was confirmed by Southern blot (Fig. 1B) or PCR analysis. The genotype ratios of the offspring from heterozygote matings were 23.6% (123 of 522) cav3–/–, 49.8% (260 of 522) cav3+/– and 26.6% (139 of 522) wild-type genotype at 4 weeks of age. Since these ratios were close to a Mendelian segregation, there was no apparent survival disadvantage of cav3–/–mice.
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) used for PCR-based genotyping. HSV-TK, herpes simplex virus thymidine kinase gene. (B) Southern blot analysis of HindIII-digested genomic DNA from mouse-tail biopsy samples obtained from littermates. As a consequence of the introduction of the HindIII site by the neo cassette, probe a was hybridized with a new 5.3 kb fragment in addition to the 13.5 kb wild-type fragment and probe b was hybridized with a new 9.2 kb fragment and the 13.5 kb wild-type fragment. +/+, wild type; +/–, heterozygous mutant; –/–, homozygous mutant. (C) Northern blot analysis. A probe specific for exon 1 of cav3 cDNA detected the transcript of correct size in 80 µg of total RNA extracted from the skeletal muscle of wild-type (+/+) and heterozygotes (+/–), but homozygous mutant (–/–) tissue showed no cav3 transcript (top). As a control for equal loading, the same membrane was rehybridized with the GAPDH cDNA probe (bottom). (D) Immunoblot analysis. Using an antibody against caveolin-3, a 20 kDa band was detected in the skeletal muscle homogenate (10, 5 and 2 µg protein/lane) of wild-type (+/+) and heterozygous mutant (+/–) mice, but not in that of homozygous mutant (–/–) mice.Analysis of gene products and caveolae
The expression of cav3 mRNA in cav3–/–, cav3+/– and wild-type mice was assessed by northern blot analysis. A probe specific for exon 1 revealed that cav3 mRNA was absent in the skeletal muscle of cav3–/– mice (Fig. 1C). In cav3+/– mice, the size of cav3 mRNA,
1.3 kb, was identical to that in wild-type mice, but its amount normalized to that of GAPDH mRNA was reduced to 47.6%.
We performed immunoblot analysis to examine caveolin-3 expression in the skeletal muscle using an anti-caveolin-3 antibody raised against an N-terminal peptide (amino acids 3–24) encoded in exon 1. Caveolin-3 was not detected in cav3–/– mice (Fig. 1D). In wild-type and cav3+/– mice, a single band with a molecular mass of 20 kDa was detected. The amount of caveolin-3 in cav3+/– mice was about half that in wild-type mice. No truncated fragments derived from the exon 2-lacking gene were detected in either mutant mice. Using the same antibody, we performed skeletal muscle immunohistochemistry. Caveolin-3 was detected in skeletal muscle plasma membrane (sarcolemma) in both wild-type and cav3+/– mice but not in cav3–/– mice (Fig. 2). The intensity of staining was weaker in cav3+/– mice than in wild-type mice. nNOS, 
-syntrophin, dystrophin, -sarcoglycan, ß-dystroglycan, sarcospan, dystrobrevin and laminin-2 were normally present in the sarcolemma of mutant mice. These proteins are known to be directly or indirectly associated with caveolin-3. Caveolin-1 was not detected in the muscle fiber of any mice, suggesting that caveolin-1 was not upregulated even when caveolin-3 was absent.
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We examined the density of caveolae in soleus muscle in cav3–/–, cav3+/– and wild-type mice using the freeze-fracture technique. As shown in Figure 3, caveolae were abundant in wild-type mice, less frequent in cav3+/– mice and scarce in cav3–/– mice. The average density of caveolae was 14.9/µm2 in wild-type, 8.0/µm2 in cav3+/– and 1.8/µm2 in cav3–/– mice (Table 1). This density in wild-type mice was comparable to the reported value in soleus muscle of B10 mice (20).
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Phenotype of cav3–/– mice
cav3+/– and cav3–/– mice grew with no clinical symptoms up to 30 weeks of age, and gained weight at a rate almost equal to that of wild-type mice, giving the same growth curves (data not shown). Neither muscle atrophy nor hypertrophy was observed.
To search for damaged muscle fibers, we injected Evans blue dye (EBD) intraperitoneally into mice between 5 and 30 weeks of age. EBD is a tracer with a small molecular mass (0.96 kDa) and tightly binds to serum albumin. Although EBD is a membrane-impermeable molecule, it is found in the cytoplasm of muscle fibers if sarcolemma integrity is compromised (21,22).
In the diaphragm of EBD-injected cav3–/– mice, a small number of EBD-containing muscle fibers with red fluorescence were recognized microscopically at 5 weeks of age (Fig. 4A, a), and many macroscopic blue lines were detected at 8 weeks of age (Fig. 4B). These lines radiated from the aortic margin to the costal margin. EBD-positive muscle fibers formed clusters microscopically (Fig. 4A, b and g). When clustered EBD-positive fibers were stained with hematoxylin and eosin (H&E), they were found to be round in shape and hypercontracted with mononuclear cell infiltration in the interstitium and muscle fibers (Fig. 4A, h). EBD-positive clusters increased in both number and size with age (Fig. 4A, c and d). Mononuclear cell infiltration and muscle fiber degeneration were still observed at 30 weeks of age. In wild-type and cav3+/– mice, no obvious EBD uptake was detected macroscopically (Fig. 4B), but a few scattered EBD-positive fibers were recognized on microscopy (Fig. 4A, e and f). Neither mononuclear cell infiltration nor muscle fiber degeneration was observed (Fig. 4A, i).
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Although no obvious EBD-positive stripes were seen macroscopically in the hindlimb muscles of cav3–/– mice up to 30 weeks of age, the soleus muscle revealed degenerating areas at 8 weeks of age on H&E staining. The degenerating areas were fairly well demarcated where muscle fibers were rounded, sometimes necrotic with macrophage invasion and interstitial tissue was edematous (Fig. 5A). Regenerating muscle fibers with centrally placed nuclei were also recognized (Fig. 5B, f). Some muscle fibers in the degenerating areas were EBD positive. The degree of degeneration of soleus muscle differed from animal to animal. The soleus muscle no longer showed such degenerating areas at 12 weeks of age when clusters of centrally nucleated muscle fibers appeared (Fig. 5B, i). Neither fibrosis nor lipid infiltration was observed. In cav3+/– mice, the soleus muscles showed no pathological changes with fiber necrosis and regeneration, except for the rarely observed round muscle fibers (Fig. 5B, b, e and h).
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In cav3–/– mice, the gastrocnemius (two of eight mice) and extensor digitorum longus (two of eight mice) muscles were also affected, but to a lesser degree than the soleus muscle. Tibialis anterior and other muscles examined exhibited no pathological changes. The cardiac muscle was not involved in cav3–/– and cav3+/– mice up to 30 weeks of age (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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We developed caveolin-3-deficient mice by targeting exon 2, which encodes the caveolin-scaffolding and membrane-spanning domains and the C-terminal region. cav3 mRNA and its protein were absent in cav3–/– mice. Both the mRNA and its protein were normal in size and their amounts were reduced by half in cav3+/– mice. No truncated mRNA or protein was detected in either mutant genotype. In Sf21 insect cells, Li et al. (7) showed that caveolin-1 or -3 expression induces the formation of caveolae, whereas caveolin-2 expression fails to induce caveolar formation. As shown in Figure 2, caveolin-1 was not expressed in the skeletal muscle fiber of any mice. The density of caveolae in cav3+/– mice was about half that of wild-type mice, whereas that in cav3–/– mice was very low. This value is comparable to the amount of caveolin-3 detected by immunoblot analysis. The finding that the density of caveolae is dependent on the amount of caveolin-3 is further supported by the recent report that transgenic overexpression of caveolin-3 causes an increase in the number of caveolae (23).
cav3–/– mice exhibited pathological lesions mainly in the soleus muscle and the diaphragm. The most notable pathological findings were muscle fiber necrosis and regeneration as seen in muscular dystrophies: muscle fiber hypercontraction, macrophage invasion, occasional edematous interstitium and regenerating fibers with basophilic cytoplasm and centrally placed nuclei. As caveolin-3 is not expressed in neurons (3), these lesions in affected muscle should not be neurogenic.
The present study indicated that the loss of caveolin-3 resulted in muscle fiber degeneration followed by regeneration, and 50% reduction was not sufficient to manifest morphological changes, suggesting that the disease is expressed in a recessive manner. We assume that pathogenic changes would not occur if caveolin-3 in the sarcolemma exceeded a certain threshold. The value should not exceed 50% of the normal amount, because pathological lesions were not detected except for the rarely observed round muscle fibers in cav3+/– mice.
According to Minetti et al. (18), the human clinical phenotype was transmitted in an autosomal dominant manner. They reported that caveolin-3 expression in the sarcolemma was considerably reduced when patients had a heterozygous mutant allele. In a separate study (24), they investigated the phenotypic behavior of wild-type and mutants of the caveolin-3 gene with the same amino acid substitution in caveolin-scaffolding and membrane-spanning domains as that observed in human patients. When the mutants were cotransfected with a wild-type gene to NIH 3T3 cells, protein products of the mutants led to the formation of unstable high molecular mass aggregates that were retained within the Golgi complex and wild-type caveolin-3 protein was not targeted in the plasma membrane. They considered that the mutants behaved in a dominant-negative fashion, causing the retention of wild-type caveolin-3 at the level of the Golgi complex. If this is the case in patients with the mutant caveolin-3 gene, it may be consistent with the autosomal dominant form of genetic transmission of caveolinopathy.
It is necessary to point out that our targeted site was exon 2, in which mutations in the patients of Minetti et al. (18) have been identified. In our cav3+/– mice, no truncated mRNA and proteins were expressed and caveolin-3 of normal size was detected in the sarcolemma. In this case, the aberrant caveolin-3, which might serve as the core of high molecular mass aggregates, was absent and normal caveolin-3 was not retained within the Golgi complex. Therefore, even in the case of humans, heterozygous individuals with a deletion of the caveolin-3 gene spanning exon 2 would lack overt symptoms and the diagnosis of caveolinopathy could not be made. This implies that a disease due to mutations of a single gene on an autosome can be inherited in both recessive and dominant manners, depending on the effects of the mutations on its protein product. Cases of single gene mutation which is transmitted in both recessive and dominant manners have been reported in nemaline myopathy with actin mutations (25).
Very recently, Galbiati et al. (23) reported that transgenic overexpression of caveolin-3 causes downregulation of dystrophin and ß-dystroglycan, leading to the muscular dystrophic phenotype. On the other hand, the same group reported that in muscle biopsies of human patients (LGMD1C) with severe caveolin-3 deficiency, dystrophin and other membrane proteins were detected at normal levels (18). In our caveolin-3-deficient mice, dystrophin and its associated proteins were immunohistochemically found to be at normal levels (Fig. 2). Thus, the molecular mechanisms of muscle degeneration may be different, although both overexpression and deficiency of caveolin-3 result in pathological changes.
Our cav3–/– mouse is a suitable animal model for studying the pathological mechanism involved in muscle fiber degeneration, although it exhibited mild degenerative processes in contrast to the naturally occurring mdx (26) and sarcoglycan-deficient mice (27–31).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Targeting-vector construction and transfection of embryonic stem cells
HindIII (13.5 kb) and KpnI (9.6 kb) mouse genomic fragments of a P1 clone containing the caveolin-3 gene (obtained from Genome Systems, St Louis, MO) were subcloned into pBluescript and characterized by restriction mapping, nucleotide sequencing and Southern blot analysis. A 4.5 kb genomic fragment as the 5' homologous region and a 6.2 kb genomic fragment as the 3' homologous region were cloned into a pPNT vector (32). The targeting vector was designed to disrupt most of exon 2 encoding the scaffolding and membrane-spanning domains and the C-terminal region of caveolin-3.
J1 embryonic stem cells were transfected with the linearized targeting vector DNA by electroporation (Gene Pulser, set at 800 V and 3.0 µF; Bio-Rad, Hercules, CA). G418 (Gibco BRL, Life Technologies, Rockville, MD) and gancyclovir (Hoffmann-La Roche, Basel, Switzerland) were added to the medium 24–48 h after the transfection for selection. A total of 336 colonies surviving G418 and gancyclovir selection were analyzed by Southern blotting of HindIII-digested genomic DNA for homologous recombination. The 5'- and 3'-flanking regions and the neomycin resistance gene were used as probes.
Generation of cav3–/– mice
Chimeric mice were generated by injecting three independent homologous recombinants with a disrupted cav3 into C57BL/6J blastocysts and then implanting them into the uteri of pseudopregnant BDF1 recipients (Clea Japan, Tokyo, Japan). Chimeric male mice were obtained and germline transmission was examined by mating them with C57BL/6J females. Agouti-coat offsprings were analyzed by Southern blot analysis. Heterozygous animals were interbred to generate homozygous mice. The genotypes were determined by Southern blot analysis or PCR on DNA obtained from a tail biopsy specimen. Wild-type and mutant alleles produced PCR products of 670 and 806 bp, respectively. Wild-type mice used in the present study were littermates of mutant mice. All animal handling procedures were in accordance with the protocol approved by the National Institute of Neuroscience (NCNP, Japan).
Northern blot analysis
Total RNA from quadriceps femoris muscles of mice was extracted using TRIzol (Gibco BRL) according to the manufacturer’s instructions. Eighty micrograms of total RNA was electrophoresed on 1.0% agarose gel containing formaldehyde and transferred to a Hybond-N+ membrane (Amersham Pharmacia Biotech, Little Chalfont, UK). Hybridization was performed as described previously (29). Mouse exon 1 sequences of cav3 cDNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA were used as hybridization probes. Blots were analyzed by a BAS5000 bio-imaging analyzer (Fuji Film, Tokyo, Japan).
Immunoblot analysis
Cryosections of quadriceps femoris muscles were boiled for 5 min in a solution containing 1% SDS and 1% ß-mercaptoethanol. After centrifugation, the protein content of supernatants was estimated using a Coomassie plus protein assay reagent (Pierce, Rockford, IL). SDS–polyacrylamide gel (14%) and the subsequent transfer to Immobilon P (Millipore, Bedford, MA) for immunoblotting were performed as described previously (33). The blots were stained using an ECL detection kit (Amersham Pharmacia Biotech, Little Chalfont, UK).
Antibodies
Rabbit polyclonal antibodies against a sequence of rat caveolin-3, TEEHTDLEARIIKDIHCKEIDL (amino acids 3–24) and against an 11.1 kDa protein fragment corresponding to the cytoplasmic domain (amino acids 1–79) on human caveolin-1 were purchased from Transduction Laboratories (Lexington, KY). A polyclonal antibody against nNOS was purchased from Zymed Laboratories (San Francisco, CA). A rat monoclonal antibody against laminin-2 (clone 4H8-2) was purchased from Alexis (Läufelfingen, Switzerland). Rabbit polyclonal antibodies against ß-dystroglycan, -sarcoglycan, sarcospan, -syntrophin and dystrobrevin were raised previously (29,34,35). An anti-dystrophin rabbit antibody was prepared against the sequence of human dystrophin, METPVTLINFWPVDSAPASS (amino acids 3406–3425). The synthetic peptide coupled with keyhole limpet hemocyaninin was injected into rabbits. Antiserum against dystrophin was purified using an affinity column coupled with the synthetic peptide.
Immunohistochemistry and histology
Thick cryosections (9 µm) of quadriceps femoris muscles from 5-week-old mice were analyzed by immunofluorescence staining using specific antibodies as described previously (33). Transverse cryosections (6–8 µm thick) of the hindlimb or diaphragm muscles of mice were processed for H&E staining as described previously (29). All sections were observed and photographed under an Olympus AX 70 microscope (Olympus Optical, Tokyo, Japan) or a laser confocal microscope SP (Leica, Wetzler, Germany).
Freeze-fracture electron microscopy
Soleus muscles were dissected from 5-week-old mice and fixed with 3% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.3. They were gradually infiltrated with glycerol up to a concentration of 30%. Freezing was carried out in liquid 2-methylbutane cooled with liquid nitrogen. The specimens were fractured at –130°C in a freeze-fracture apparatus (BAF060; Bal-Tec, Balzers, Liechtenstein) at a vacuum of 3.6 x 10–5 to 1 x 10–4 Pa, followed by replication with platinum and carbon. The replicas were examined under an electron microscope (JEM1010; JEOL, Tokyo, Japan).
Vital staining with EBD
To detect damaged muscle fibers, EBD (10 mg/ml in phosphate-buffered saline) was injected intraperitoneally into mice (0.1 ml/10 g body wt) as described previously (29). The mice were sacrificed 20 h after the injection, and their muscles were sectioned and examined under a fluorescence microscope (AX 70; Olympus Optical).
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ACKNOWLEDGEMENTS |
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We would like to thank Drs Akio Takahashi and Tetsuya Matsuzaki for managing our mouse facility, Dr Richard Mulligan for the pPNT vector, Dr En Li for J1 ES cells and Ms Masako Kitahara for technical assistance. This work was supported in part by a Research Grant (11B-1) for Nervous and Mental Disorders from the Ministry of Health and Welfare, Japan, and a Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Science, Sports and Culture, Japan.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +81 42 341 2711; Fax: +81 42 346 1746; Email: hagiwara@ncnp.go.jp
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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