1 First Department of Internal Medicine and 4 Central Research Laboratory, Osaka Medical College, Takatsuki-city, Osaka 569-8686; 2 Department of Pharmacology, Development Research Laboratories, Kaken Pharmaceutical Company, Yamashina-ku, Kyoto 607; and 3 First Laboratory for Pharmacological Research Institute for Life Science Research, Asahi Chemical Industry Company, Tagata-gun, Shizuoka 416-0934, Japan
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ABSTRACT |
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Migration and proliferation of smooth muscle cells (SMC) contribute to neointimal formation after arterial injury. However, the relation between migration and proliferation in these cells is obscure. To discriminate between migration and proliferation, we employed a migration assay of SMC at different phases of the cell cycle. Serum-deprived SMC were synchronized in different phases of the cell cycle by addition of serum for various periods of time. Migration induced by platelet-derived growth factor B-chain homodimer was maximal in SMC that were predominantly in the late G1 (G1b) phase. In addition, in nonsynchronized SMC, 65-75% of SMC that had migrated were in the G1b phase. Phosphorylated myosin light chain was enriched around the cell periphery in SMC in the G1b phase compared with SMC in the other cell cycle phases. Interestingly, the Triton X-100-insoluble fraction of myosin was remarkably decreased in G1b-enriched SMC. These findings suggest that migratory activity of SMC may be coupled with the G1b phase. The phosphorylation and retention of myosin might explain some of the properties responsible for increased migration.
myosin; atherosclerosis
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INTRODUCTION |
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MIGRATION AND PROLIFERATION of smooth muscle cells (SMC) are the major causes of the early phase of atherosclerosis and restenosis after coronary angioplasty (8, 30, 42). Initiation of both of these processes is mediated primarily by a number of regulatory polypeptides, such as platelet-derived growth factor (PDGF) (8, 30). Both proliferation and migration of arterial SMC are markedly stimulated by the PDGF B-chain homodimer (PDGF-BB), and inhibition of PDGF in vivo partially blocks SMC accumulation after balloon injury of normal vessels (8). However, the relation between proliferation and migration is obscure.
Proliferating cells exhibit a wide range of morphological and physiological properties throughout the cell cycle. As the cell cycle progresses, an increase in cell volume is accompanied by an increase in cell viscosity. Before mitosis, anchor-dependent cells round up and become less adhesive to their neighbors. The cell cycle is coupled to the organization of the cytoskeleton and cell-cell and cell-extracellular matrix interactions (13). We reported that overexpression of the cyclin-dependent kinase inhibitor p21Cip1, which induces cell cycle arrest, inhibited SMC migration (9).
Moving cells exhibit actin-rich-leading lamellipodia in which actin filaments undergo rapid turnover (6, 41); relatively stable actin-myosin cables are present near the middle and in the rear of the cell as well as in more established protrusions (46). Myosin II forms discrete clusters of bipolar minifilaments in lamellipodia that increase in size and density toward the cell body boundary and colocalize with actin in boundary bundles (5). Knockout of myosin II in Dictyostelium results in a dramatic decrease in the rate of cell locomotion (44) and in blockade of locomotion in an environment of increased resistance (7, 14). Thus myosin II is thought to play a major role in the regulation of cell shape and the direction and rate of cellular migration (28, 39).
The mechanisms for cell migration are contraction of microfilament bundles organized in a sarcomeric-like fashion (18, 34) and myosin-driven transport of cell body components along uniformly polarized actin arrays (24). Myosin light chain (MLC) phosphorylation is an important process in cell contraction (23, 40). Motile fibroblasts with a polarized cell shape exhibit a bimodal distribution of phosphorylated MLC along the direction of cell movement (22).
Here, we show for the first time the relation between cell cycle in the late G1 (G1b) phase and SMC migration. Our data suggest that myosin light chain monophosphorylation at serine 19 (MLC-P) may play a critical role in SMC migration during the G1b phase.
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METHODS |
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Antibodies.
Monoclonal antibodies were used to detect paxillin (Chemicon),
phosphotyrosine (PY20; Transduction Laboratories), and human -actin
(1A4) and nonphosphorylated and phosphorylated MLC (Sigma Chemical). To
detect the MLC-P of smooth muscle, a specific antibody was raised
against the phosphorylated synthetic peptide
Lys-Lys-Arg-Pro-Gln-Arg-Ala-Thr-phosphoSer-Asn-Val-Phe-Cys. This
antibody recognizes only the MLC-P, i.e., with no detectable recognition of nonphosphorylated or diphosphorylated MLC (32, 33). Polyclonal antibodies were used to detect focal
adhesion kinase (FAK) (C-20; Santa Cruz).
Cell culture.
Human aortic SMC (passage 5 or 6) were obtained
from Clonetics (San Diego, CA) and cultured in modified MCDB131 medium
supplemented with 5% fetal calf serum, 100 U/ml penicillin, 100 mg/ml
streptomycin, 10 ng/ml epidermal growth factor, 2 ng/ml basic
fibroblast growth factor, and 1 mmol/l dexamethasone (27).
The SM3 cell line, a strain of rabbit aortic arterial SMC, was
characterized by biochemical, histochemical, and pharmacological traits
as being reversible between contractile and synthetic phenotypes
(33, 35). SM3 cells were positive for staining of
-actin and calponin. In addition, cells serum deprived for 2 days
contracted in response to stimulation by PGF2
and
endothelin (35).
Indirect immunofluorescence. SMC were plated on 8-mm2 Lab-Tek chamber slides (Nunc) coated with 20 µg/ml human fibronectin (Sigma Chemical). Cells to be stained were fixed with 2% paraformaldehyde for 20 min at room temperature, permeabilized with 0.5% Triton X-100 (Triton), and blocked by 10% goat serum diluted with PBS for 60 min at room temperature. The specific MLC-P antibody was diluted with 2% goat serum in PBS (1:60) and then incubated overnight at 4°C. The specimens were then incubated with a rhodamine-conjugated secondary antibody (Molecular Probes) mixture containing FITC-phalloidin (Sigma) for 1 h at room temperature. Finally, the slides were observed through a fluorescence microscope (Olympus). To estimate the number of cells that have peripheral staining of MLC-P, we counted 300 cells in each of the cell cycle phase-enriched populations and then calculated the ratio of the cells showing peripheral staining.
Cell cycle staining. Cells were stained according to the method of Darzynkiewicz et al. and Yamamoto et al. (4, 17, 45). Briefly, cells were trypsinized, fixed with 70% ethanol at 4°C for at least 60 min, and rinsed with PBS. The cell pellets (1 × 106 cells) were suspended in 0.2 ml of a cold solution containing 0.1% Triton, 0.08 N HCl, and 0.146 M NaCl and then incubated for 1 min on ice. The cells were then stained by incubation at room temperature for 5 min with dye solution containing 14 µg/ml acridine orange (AO; Molecular Probes), 1 mM EDTA, 0.146 M NaCl, and 0.1 M citrate phosphate buffer (pH 6.0). After flow cytometry, the cells were shown microscopically to be completely intact, as were the control cells without detergent treatment.
Flow cytometry. Cell fluorescence was measured on a flow cytometer (Elite, Coulter Electronics) using an argon ion laser (488 nm) at a flow rate of 1,000-1,200 cells/s (45). The red (630 nm) and green (525 nm) fluorescence emissions from each cell were separated optically and quantitated by separate photomultipliers. Background fluorescence was automatically subtracted. The specificity of detection of the DNA and RNA contents was evaluated by preincubation of permeable cells with DNase I (1 mg/ml, Sigma) and RNase A (1 mg/ml, Sigma). The amount of red and green fluorescence was proportional to the amount of RNA and DNA per cell, respectively. The early G1 (G1a) phase of the cell cycle involves an increase in the levels of RNA and protein in preparation for entry into the G1b and S phases. Cells in the G1a phase contain higher amounts of RNA than quiescent cells in the G0 phase. The G1b phase is defined as the stage when RNA levels are equivalent to those seen in early S phase before DNA synthesis (4, 17, 45).
Adhesion assay. An adhesion assay was performed as described previously (9). Briefly, a 96-well plate was coated with 20 µg/ml human fibronectin (Sigma Chemical) or 300 µg/ml bovine collagen type I (Chemicon) and blocked with 10 mg/ml bovine serum albumin. The cells were trypsinized, and 2.0 × 104 cells were added to each well. After 1 h of incubation at 37°C, the cells were washed twice with PBS, fixed in 4% paraformaldehyde for 10 min, stained with 0.5% toluidine blue, and rinsed in water. Cells were solubilized by the addition of 100 µl of 1% SDS and quantified using a microtiter plate reader at 595 nm. The experiments described were repeated a minimum of three times.
Migration assay. Migration was assayed by a modification of the Boyden's chamber method using microchemotaxis chambers (Neuroprobe) and polycarbonate filters (Nucleopore) with a pore size of 5.0 µm, as described previously (9). Briefly, the filters were coated with 20 µg/ml fibronectin or with 300 µg/ml collagen I and placed between the upper and lower chambers. Cells were trypsinized and suspended at a concentration of 5.0 × 105 cells/ml in MEM supplement. The SMC (2.5 × 104 cell in 50 µl) were placed in the upper chamber, and 25 µl of MEM containing human recombinant PDGF-BB (Sigma Chemical) were placed in the lower chamber. The chamber was incubated at 37°C in 5% CO2 for 3 h. The filter was removed, and the nonmigrating SMC, remaining on the upper side of the filter, were scraped off. SMC that had migrated to the lower side of the filter were fixed in methanol, stained with Diff-Quick staining solution, and counted in five fields under a microscope (×200) to quantify SMC migration. Migratory activity was expressed as the mean number of cells that had migrated to the lower side of the filter. The experiments described were repeated a minimum of three times.
Separation of cells that had migrated. Nonsynchronized cells were induced to migrate by PDGF-BB with the use of microchemotaxis chambers. Cells that had migrated were collected and stained to determine the relative distribution among the phases of the cell cycle as described in Flow cytometry. The SMC from the lower side of the filter were trypsinized and fixed with 70% ethanol at 4°C for 60 min. A minimum of 5,000 cells were collected for each experiment. Cells were examined for their relative distribution among the phase of the cell cycle as described in Flow cytometry.
Measurement of MLC phosphorylation. Cells were collected by trypsinization and allowed to spread on fibronectin-coated plastic dishes for 1 h at 37°C. The cells were then frozen by immersion in 10% trichloroacetic acid (TCA) containing 10 mM dithiothreitol (DTT) cooled by dry ice. Frozen cells were washed twice with acetone containing 10 mM DTT to remove the TCA and dried. The dried cells were resuspended in 8 M urea sample buffer, electrophoresed, and transferred to the nitrocellulose membrane as described previously (37). The nitrocellulose membrane was incubated overnight with 10 µg/ml of anti-MLC IgG. The region containing 20-kDa MLC (MLC20) was visualized as dark blue bands, using a Vectastain ABC kit (Vector, Burlingame, CA) and 4-chloronaphthol as the substrate for peroxidase. Densitometry of immunoblots and quantitation of absorbance peaks were performed with a Densitron PAN-FV (Jookoo, Tokyo, Japan) equipped with a recording integrator. The extent of MLC20 phosphorylation is expressed as the percent MLC in the monophosphorylated or diphosphorylated forms, respectively.
Measurements of FAK and paxillin phosphorylation. Cells were collected by trypsinization and allowed to spread on fibronectin-coated plastic dishes for 1 h at 37°C. The cells were resuspended in ice-cold lysis buffer: 50 mM HEPES (pH 7.5), 50 mM NaCl, 5 mM EDTA, 1% Triton, 50 mM NaF, 3 µg/ml leupeptin, 3 µg/ml aprotinin, 1 mM Na3VO4, and 1 mM phenylmethylsulfonyl fluoride. Protein concentrations were measured by the Bradford method (Bio-Rad). Paxillin and FAK were immunoprecipitated from 35 µg of lysate protein with 1 µl of the monoclonal antibody against FAK and paxillin. Immunoprecipitates were divided in two, separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with the anti-phosphotyrosine antibody (1:500). Alkaline phosphatase-conjugated secondary antibodies were used in the detection system. The experiments described were repeated a minimum of three times.
Quantification of Triton-insoluble pools. Triton-insoluble proteins were determined as cytoskeletal-associated proteins by a modification of the technique of Watts and Howard (43). Cells were lysed with 1% Triton X-100 buffer (10 mM imidazole, 40 mM KCl, and 10 mM EGTA, pH 7.15, with 7 mM diisopropyl fluorophosphate to prevent proteolysis), and equal amounts of protein determined by the Bradford method were centrifuged (36,000 g for 5 min). The pellets were separated from a Triton-soluble fraction and solubilized in a Tris buffer (0.625 M in 2% SDS, 10% glycerol, and 5% 2-mercaptoethanol), electrophoresed on a 12% SDS-gel, transferred, probed with primary antibodies, and visualized with alkaline phosphatase-conjugated secondary antibodies. The experiments described were repeated a minimum of three times.
Statistical analysis. Data are presented as means ± SE. Differences between groups were calculated by the Student's t-test. A value of P < 0.05 was considered significant.
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RESULTS |
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Scattergrams of DNA and RNA.
The changes in DNA and RNA histograms for SMC stained with AO were
studied by flow cytometry. Human SMC and SM3 cells deprived of serum
for more than 36 h were stimulated with readdition of serum for
various periods of time. The cells were stained with AO and were
represented on the two-dimensional scattergrams in which individual
cells were plotted on the basis of their respective levels for red (630 nm) and green (525 nm) fluorescence. Table 1 shows different cell cycle phases after
addition of serum to human SMC. Each cell cycle phase (mainly in
G1a, G1b, S, and G2/M) was induced by serum
incubation for 0, 14, 25, 34 h, respectively (Table 1).
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Cell cycle phase effects on cell migration.
To examine the effect of cell cycle state on cell migration, the cells
of different cell cycles were tested for migration using the modified
Boyden chamber assay. PDGF-BB stimulated migration and chemotaxis of
SMC in a dose-dependent manner at concentrations up to 30.0 ng/ml on
fibronectin-coated and collagen I-coated polycarbonate filters (Fig.
1). A large number of human SMC with 14-h
serum addition were in G1b phase (Table 1), and their
migration was the highest of all on both fibronectin and collagen I at
every PDGF concentration (Fig. 1). The absence of PDGF-BB can induce migration at this time point. In addition, serum-induced migration was
also the highest in G1b-rich human SMC (data not shown).
The similar results were seen in SM3 cells (data not shown). These findings suggested that the increase in migration of SMC might be
related to G1b phase.
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Differences in migration activity independent of cell adhesion.
Cell adhesion to a substratum is thought to be an important process in
cell migration (18). The various synchronous human SMC and
SM3 cells (Table 3) were tested for
strength of adhesion to fibronectin and collagen I. The cells attached
to these substrates were counted by absorbance 1 h after delivery
of the cell suspension. However, there was no difference in number of
attached cells among the various synchronous human SMC (Fig.
2, A and B) and SM3
cells (Fig. 2, C and D).
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Increase of phosphorylated myosin in cell periphery.
Cell migration speed is thought to be related to the contractile force
needed to move the cell body forward (19). MLC
phosphorylation is one of the most important mechanisms involved in the
contraction of smooth muscle (40). Attachment on
fibronectin with PDGF-BB induced rapid filament conformation of MLC-P
in SMC (19, 33). To examine the reasons for differences in
migratory activity, we examined the subcellular localization of MLC-P
of SM3 cells using a specific antibody (33). Because SM3
cells are well characterized in the phosphorylation of MLC (33,
37), we used these to analyze the MLC-P. Figure
4 shows the double-labeled
immunolocalization of MLC-P and F-actin in SM3 cells attached to
fibronectin for 1 h with 6 ng/ml PDGF-BB. Dramatically, MLC-P
appears to be enriched in the cell edge and around the nucleus in
one-third of SM3 cells with 14 h of serum addition (Fig.
4D). This staining pattern was seen in 7.4, 33.8, 7.6, and 7.3% of SM3 cells with 0, 14, 22, and 30 h of serum
addition, respectively.
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Quantitative analysis of Triton-insoluble myosin.
To characterize and define the contribution of altering the
distribution of MLC-P, we examined the effect of ionic strength on
retention of myosin in the Triton-insoluble pool. SM3 cells were
attached to fibronectin for 1 h with 6 ng/ml PDGF-BB and then
treated with 1% Triton. The Triton-insoluble pools were separated from
Triton-soluble pools and studied. MLC in the Triton-insoluble pool was
markedly decreased in SM3 cells with 14 h of serum addition, although the total amounts of MLC (Triton-soluble plus Triton-insoluble pool) in the cells were not different (Fig.
5). However, the Triton-insoluble F-actin
level was similar (Fig. 5).
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DISCUSSION |
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SMC migration is linked to cell cycle phase. In this study, we demonstrated for the first time that G1b phase SMC underwent significant PDGF-BB and serum-induced migration and chemotaxis. In the rat model, the first response to balloon injury, called first wave, consists of medial SMC replication (36). Although the first wave has no obvious relation to the later migration and intimal proliferation, several studies with antisense agents directed at cell cycle genes have shown that inhibition of these initial proliferative events leads to a diminution of the final extent of neointimal proliferation (25, 26). Furthermore, we previously reported that overexpression of the cyclin-dependent kinase inhibitor p21Cip1, which induces cell cycle arrest, inhibited SMC migration (9). Thus we speculated that cell cycle phases were associated with cell migration. We showed that migratory activities of human SMC and SM3 cells, which have cell cycles mainly in G1b phase, were significantly increased on fibronectin and collagen I. In addition, in nonsynchronized human SMC and SM3 cells, 65.8-75.8% of PDGF-BB-induced migrated cells were in the G1b phase (Table 2). These findings indicate that SMC of G1b phase exhibit greater migration on fibronectin and collagen I.
Clowes et al. (3) reported that SMC unlabeled by [3H]thymidine, called nondividing cells, could migrate and contribute to the increase in neointimal formation in the rat ballooned carotid (3). According to the present study, intimal SMC unlabeled by [3H]thymidine might to be in the G1b phase. For endothelial cells, migration is observed as early as 4 h after scraping of a confluent cell monolayer; however, significant incorporation of [3H]thymidine dose not occur until 12-20 h after scraping (1, 38). Furthermore, for lymphocytes, an increase in lymphocyte migration occurs in G1 phase but not in S or G2/M phase (21, 29). These findings are consistent with our results. Together, we speculate that medial SMC in vivo migrating toward the intima in second wave may be associated with the G1b phase of their cell cycle. Thus inhibition of transition from G1a to G1b may regulate not only cell proliferation but also cell migration from media to intima, and it may be the therapeutic point for inhibition of neointimal formation.Increase in migration is independent of cell adhesion to substratum. To examine the reasons for differences in migratory activity, cell adhesion to substrata was examined. However, no differences in adhesion strength (Fig. 2) and phosphorylation level of FAK and paxillin (Fig. 3) were found among cell cycle states. These findings suggest that cell cycle-dependent migration has little influence on cell adhesion and that different migratory activity may be differentially regulated. In fact, mitogen-activated protein (MAP) kinase increases cell migration in an adhesion-independent manner (15).
Different localization of MLC-P in G1b cells. Translocation of the cell body forward, once the membrane protrusion has become adherent to the substratum, may occur by myosin interactions with actin filaments, the contraction of filaments connecting cell-substratum adhesion complexes with intracellular structures, or relative movement of adhesion complexes across cortical actin filament tracks (19). Myosin II, the only member of the myosin superfamily with the ability to form polymeric supramolecular assemblies (2), has clearly been shown to participate in the overall process of cell motility. Although it has been suggested that protrusion of the leading edge is responsible for cell migration, it is still unclear how myosin II is involved in cell motility.
Analysis of the organization and dynamics of myosin with respect to actin is needed to test these hypotheses and to determine the mechanism of cell body translocation. To elucidate this mechanism, we examined the supramolecular organization of the actin-myosin II system and the dynamics of phosphorylated MLC in SMC. Immunocytochemistry with specific antibody against MLC-P demonstrated that MLC-P is enriched in the cell edge and around the nucleus in one-third of G1b phase-enriched SM3 cells (Fig. 4). MLC-P forms a ring or arc at the margin of these cells. The high level of MLC-P in the peripheral microfilament bundles was reported in the moving edge of epithelial cells and SM3 cells (22, 33). These were consistent with our result that one-third of G1b phase-enriched cells expressed MLC-P in the cell edge. However, the total level of MLC-P was similar in these cells (Table 4), although we cannot rule out the possibility that the total level of MLC-P of each cell might be different. To analyze the contribution of altering the distribution of MLC-P, we examined the retention of myosin and actin in the Triton-insoluble pool. The simplest interpretation is that soluble myosin consists of free monomers, and cytoskeletal myosin is assembled into thick filaments and/or bound to actin microfilaments. The cytoskeletal myosin is released by addition of MgATP (16), and the released myosin is soluble in Triton. We separated the Triton-insoluble pools from Triton-soluble pools. Although levels of Triton-insoluble F-actin on immunoblots were similar (Fig. 5), MLC in the Triton-insoluble fraction was remarkably decreased in G1b phase-enriched SM3 cells compared with SM3 cells in the other cell cycle phases. The assembly-disassembly occurs to some extent during a contraction-relaxation cycle in smooth muscle (10), especially in moving cells. Together with data in Fig. 5, the amount of free monomers of myosin appears to increase in G1b phase-enriched SM3, which might be associated with cell migration.Intracellular signaling pathways of increased migration. Cellular recognition of growth factors and adhesive proteins influences proliferation, differentiation, and migration. The Ras/MAP kinase signaling pathway leads not only to transcriptional control of cell proliferation and differentiation but also to cell migration by directly impacting the machinery (12, 15). MAP kinase increases cell migration but not adhesion or spreading on collagen (15) in the same manner as that described in the present study. MLC kinase has been identified previously as an MAP kinase substrate that may be involved in stimulating cellular migration (15). These results may imply that a high level of MLC-P in the peripheral cell edge in G1b phase-enriched cells is associated with activation of MLC kinase and MAP kinase signal (11). Further studies are required to clarify this.
The findings of the present study may explain the importance of cell cycle state in SMC migration and suggest that localization of MLC-P may play a critical role in the SMC migration during the G1b phase. Thus, in these findings, it is suggested that the G1a/G1b transition of SMC cell cycle is not only carried out on the SMC proliferation in atherosclerosis and restenosis but also on the SMC migration. Furthermore, G1a/G1b transition of SMC cell cycle or inhibition of MLC phosphorylation may be a new therapeutic point for atherosclerosis and restenosis. ![]() |
ACKNOWLEDGEMENTS |
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We thank Drs. Kristine E. Kamm and Robert Grange (The University of Texas Southwestern Medical Center) for excellent comments concerning the mechanisms of cell migration. We also thank Dr. K. Yamamoto (Tokyo Metropolitan Institute of Gerontology) for technical advice of flow cytometry, Dr. K. Sakurada (Asahi Chemical Industry) for technical advice on immunocytochemistry, and Dr. Eiji Isotani (The University of Texas Southwestern Medical Center) for technical support for measurement of MLC phosphorylation.
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FOOTNOTES |
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Address for reprint requests and other correspondence: R. Fukui and M. Hoshiga, First Dept. of Internal Medicine, Osaka Medical College, 2-7 Daigaku-cho, Takatsuki-city, Osaka 569-8686, Japan (E-mail: in1038{at}poh.osaka-med.ac.jp).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 29 July 1999; accepted in final form 21 April 2000.
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