Molecular Human Reproduction, Vol. 7, No. 10, 979-985,
October 2001
© 2001 European Society of Human Reproduction and Embryology
Implantation and pregnancy |
Nitric oxide increases matrix metalloproteinase-1 production in human uterine cervical fibroblast cells
1 Department of Gynecology and Obstetrics, Kyoto University Graduate School of Medicine, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, 2 Pharmacology Group, R&D Administration and Coordination, Research & Development Division, Nippon Organon K.K., 1-5-90 Tomobuchi-cho, Miyakojima-ku, Osaka 534-0016 and 3 Department of Biochemistry, Tokyo University of Pharmacy and Life Science, Horinouchi, Hachioji, Tokyo 192-0392, Japan
Abstract
Since uterine cervical ripening is an active biochemical process similar in part to an inflammatory reaction, nitric oxide (NO) has been proposed as a key mediator of this event. However, the mechanism by which NO modulates human cervical ripening has not been fully elucidated. In the present study we investigated the presence of NO synthases in human uterine cervix by immunohistochemistry and reverse transcriptase–polymerase chain reaction analysis. Furthermore, we examined the presence of NO-mediated regulation of matrix metalloproteinase-1 (MMP-1) production in cultured human uterine cervical fibroblast cells using enzyme-linked immunosorbent assay and Northern blot analysis. Endothelial and inducible NO synthases were detected in the form of mRNA and protein expression in pregnant uterine cervix. Interleukin-1
(IL-1) increased the expression of inducible NO synthase mRNA in cultured human uterine cervical fibroblast cells. IL-1 also increased MMP-1 secretion from the cultured cervical fibroblast cells. This IL-1-augmented MMP-1 secretion was partially but significantly blocked by an NO synthase inhibitor, NG-nitro-L-arginine methyl ester. On the other hand, NO donors increased MMP-1 production in the cultured cervical fibroblast cells. These findings together suggest that NO contributes to the process of cervical ripening via enhancement of MMP-1 production, and that IL-1 increases MMP-1 secretion from cervical fibroblasts at least in part via NO synthesis.
interleukin-1/matrix metalloproteinases/nitric oxide/nitric oxide synthase/uterine cervical ripening
Introduction
Uterine cervical ripening and dilatation are essential events in a non-complicated vaginal delivery. Uterine cervical ripening is an active biochemical process similar in part to an inflammatory reaction, and involves a complex cascade of sequential reactions, including degradation of extracellular matrix proteins and glycoproteins, disruption of tightly aligned collagen fibrils, and increase in hydration caused by hyaluronan (Junqueira et al., 1980
; Uldbjerg et al., 1983a; El Maradny et al., 1997).
The central event in the complicated process of cervical ripening is the degradation of collagens, since the human uterine cervix is composed mainly of fibroblast cells and fibrous connective tissues in which collagen and glycosaminoglycans predominate (Rorie and Newton, 1967; Uldbjerg et al., 1983b). Accordingly, collagenases, recently termed matrix metalloproteinases (MMP), are believed to be the key enzymes in the degradation of collagens during cervical ripening (Ito et al., 1991, 1998; Imada et al., 1997). The MMP are a family of at least 16 different types of zinc-dependent enzymes, capable of degrading collagen as well as other extracellular matrix components such as proteoglycans, fibronectin and laminin present in the interstitial matrix and basement membranes (Hulboy et al., 1997; Konttinen et al., 1999). Each MMP has substrate selectivity. The major extracellular matrix proteins in the human uterine cervix are collagen type I and type III (Kleissl et al., 1978; Minamoto et al., 1987) which are the preferred substrates of MMP-1 (Hulboy et al., 1997). Thus, it is plausible that MMP-1 is involved in the degradation of collagens during the process of cervical ripening. Indeed, MMP-1 mRNA expression has been reported in cultured human uterine cervical fibroblast cells (Sugano et al., 2000) and in cultured human uterine cervical smooth muscle cells (Watari et al., 1999).
Nitric oxide (NO), originally identified as a potent vasodilative substance, has been reported to contribute to the maintenance of pregnancy, by allowing myometrial relaxation and an increase in uterine blood flow during pregnancy (Weiner et al., 1994; Kublickiene et al., 1997; Magness et al., 1997). We have previously reported that maternal plasma NO metabolite concentrations increase during pregnancy, and that the maternal plasma concentration of cyclic guanosine 3',5'-monophosphate (cGMP), a second messenger of NO, is increased in women with mild pre-eclampsia, supporting the possibility that NO plays physiological roles in pregnancy (Itoh et al., 1997; Nanno et al., 1998)
Nitric oxide is also involved in inflammatory reactions (Cohen et al., 1998). The induction of inducible nitric oxide synthase (iNOS) can generate a sustained elevation of NO concentrations which may exert various pro-inflammatory effects, including vasodilation, oedema, cytotoxicity, tissue remodelling, and various other cytokine-dependent processes. Since uterine cervical ripening is an active biochemical process in some respects similar to an inflammatory reaction (Junqueira et al., 1980), the possible involvement of NO in cervical ripening has been proposed (Calder, 1998; Chwalisz and Garfield, 1998; Norman et al., 1998). Indeed, the augmentation of iNOS mRNA expression has been reported in the process of uterine cervical ripening in rats and humans (Ali et al., 1997; Tschugguel et al., 1999; Ledingham et al., 2000). Moreover, it has been reported that in-vivo treatment with NO donors causes uterine cervical ripening in rats, guinea-pigs and humans (Chwalisz et al., 1997; Thomson et al., 1998; Shi et al., 2000).
Thus, NO may be one of the more important modulators in the process of cervical ripening. However, Ledingham et al. have reported that NO donors do not alter MMP-2 or MMP-9 secretion from cultured human uterine cervical fibroblast cells (Ledingham et al., 1999). Thus, as far as we are aware, the mechanism by which NO induces uterine cervical ripening has not been fully elucidated. To clarify the possible effect of NO on the degradation of uterine cervical collagens, we investigated (i) whether iNOS and endothelial nitric oxide synthase (eNOS) are expressed in the uterine cervix; (ii) whether interleukin-1 (IL-1), one of the key modulators of cervical ripening (Garcia-Velasco and Arici, 1999), augments iNOS mRNA expression in cultured uterine cervical fibroblast cells; and (iii) whether NO increases MMP-1 production in cultured cervical fibroblast.
Materials and methods
Reagents
All reagents were of analytical grade and purchased from Sigma Chemical Co. (St Louis, MO, USA), unless otherwise indicated.
Materials
Uterine cervical tissues were obtained with written informed consent from pre-menopausal non-pregnant (n = 5) and pregnant women (n = 7) who had undergone total hysterectomy for gynaecological reasons such as uterine cervical cancer (7, 9 and 11 weeks of gestation) ovarian cancer (12 weeks of gestation), and uterine leiomyoma (n = 8: five non-pregnant women in the early follicular phase and three women at 10, 10 and 11 weeks of gestation). Tissues were snap-frozen by liquid nitrogen and kept at –80°C until assayed. The experimental protocol was approved by our institutional ethical committee.
Preparation of cultured human uterine cervical fibroblast cells (CxF cells)
Cervical stromal tissues were obtained from non-pregnant women (n = 3) and first trimester pregnant women (n = 4; 9, 10, 10 and 11 weeks of gestation), rinsed several times in 10 mmol/l phosphate-buffered saline and minced into ~2 mm pieces, which were placed on 6 cm collagen-coated culture dishes and gently covered with a thin micro cover glass. Cells were cultured in Minimum Essential Medium (MEM) (Gibco BRL, Rockville, MD, USA) containing 10% fetal bovine serum, at 37°C in 95% air and 5% CO2 under humidified conditions. The cells from the tissues were grown to passage 6, and then used as CxF cells. At each passage, an aliquot of the cells was placed on several Neoprene-coated slide glasses (Nissin EM, Tokyo, Japan) and incubated for 24 h, and then fixed in acetone at –20°C for 5 min. The slides were incubated with anti-cytokeratin (1:100; Dako Co., Carpinteria, CA, USA), anti-vimentin (1:100; Dako), or anti--smooth muscle actin (1:100; Dako) monoclonal antibodies for 30 min at room temperature. After a 30 min incubation with fluorescein isothiocyanate-conjugated anti-mouse IgG rabbit serum as a second antibody (40 µg/ml; Dako), the slides were mounted with Perma Fluor Aqueous Mounting Medium (Immunon, Pittsburgh, PA, USA), and examined under a fluorescence microscope by two individuals.
The immunofluorescence study showed 99% positive staining for vimentin in cervical fibroblast cells at the sixth passage (Figure 1A), and <1% positive staining for both cytokeratin (Figure 1B) and -smooth muscle actin (Figure 1C), indicating a high purity of cervical fibroblast cells. A negative control using normal mouse IgG showed minimal staining (Figure 1D). Thus, the cervical fibroblast cells at the sixth passage were confirmed to be of high purity. These cells were therefore used in the further experiments in this study.
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Experimental protocol
When CxF cells at the sixth passage reached confluence, the medium was replaced with fresh MEM without serum. Subsequently, the cells were further incubated for 24 h in 12-well or 10 cm collagen-coated plates in the presence or absence of several substances. The culture medium was then collected and used for the MMP-1 enzyme-linked immunosorbent assay (ELISA) and the cells were used for reverse transcriptase–polymerase chain reaction (RT–PCR) and/or Northern blot analysis. The substances tested were sodium nitroprusside (SNP; a NO donor, 10–7–10–4 mol/l), diethylenetriamine nitric oxide (DETA-NO; another NO donor, 10–6–10–4 mol/l), 8-bromo-1,2-guanosine cyclic monophosphate (8-bromo-cGMP; a membrane-permeable derivative of the NO second messenger, cGMP, 5x10–5–5x10–3 mol/l), NG-nitro-L-arginine methyl ester (L-NAME; an inhibitor of NOS, 10–20 mmol/l), and interleukin-1
(IL-1, 1.0 ng/ml; a kind donation from Daiichi Pharmaceutical Co. Ltd, Tokyo, Japan). All media samples were kept at –20°C until assayed.
Immunohistochemical analysis of iNOS and eNOS expression
Uterine cervical specimens were embedded in OCT compound (Sakura Finetek Inc., Torrance, CA, USA), and stored at –80°C. The sections were incubated for 1 h at room temperature with anti-iNOS monoclonal antibody (1.25 µg/ml) or anti-eNOS monoclonal antibody (2.5 µg/ml) (Transduction Co, Lexington, KY, USA). Staining was performed using a biotinylated horse anti-mouse IgG as the secondary antibody and the avidin–biotin–peroxidase (ABC) method (Elite ABC; Vector Laboratories, Burlingame, CA, USA) with 3,3'-diaminobenzidine (DAB).
RT–PCR analysis of iNOS and eNOS mRNA expression
To examine the expression of iNOS and eNOS, RT–PCR analysis was carried out. Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) gene expression was also measured by RT–PCR as a control. Total RNA was extracted as previously described (Masuzaki et al., 1997). After reverse transcription of 2 µg of total RNA from human pregnant or non-pregnant uterine cervix or CxF cells using oligo(dT) primer (Promega, Madison, WI, USA) and SuperscriptTM II (Gibco BRL, Rockville, MD, USA), the resulting single-stranded cDNA was subjected to PCR. Forward and reverse primers used for amplification of the human iNOS (Chartrain et al., 1994) and eNOS coding regions (Nadaud et al., 1994) were: iNOS forward: 5'-GAGCTTCTACCTCAAGCTATC-3'; iNOS reverse: 5'-CCTGATGTTGCCATTGTTGGT-3'; eNOS forward: 5'-GCACAGGAAATGTTCACCTAC-3'; and eNOS reverse: 5'-CACGATGGTGACTTTGGCTAG-3'. Forward and reverse primers for the human G3PDH coding region were purchased from Clontech Laboratories, Inc. (Palo Alto, CA, USA). The PCR was performed using TaKaRa TagTM (Takara Shuzo Co. Ltd, Otso, Shiga, Japan) according to the manufacturer's instructions. The reaction mixture (100 ml) consisted of TaKaRa TaqTM (2.5 units), 10 mmol/l Tris–HCl (pH 8.3), to 50 mmol/l KCl, 1.5 mmol/l MgCl2 and 0.2 mmol/l dNTP. The expected sizes of final products from human iNOS and eNOS cDNA were 312 and 645 bases respectively. The PCR programme used was amplification for 32 cycles of 30 s at 94°C, 60 s at 55°C, and 60 s at 72°C. Final products were extended to full length by incubation for 4 min at 72°C.
Quantitative RT–PCR of human iNOS and G3PDH mRNA was carried out using real time TaqmanTM technology and analysed using a Model 7700 Sequence Detector (Perkin Elmer Applied Biosystems, Wellesley, MA, USA) (Heid et al., 1996). Forward and reverse primers and Fam/Tamra- or Joe/Tamra-labelled probes used for amplification of part of the human iNOS (Chartrain et al., 1994) and G3PDH coding regions (Tso et al., 1985) were: iNOS forward: 5'-GGCTGCCAAGCTGAAATTGAAT-3'; iNOS reverse: 5'-CGTGATAGCGCTTCTGGCTCTT-3'; iNOS (Fam/Tamra) probe: 5'-Fam-AGGAGCAGGTCGAGGACTATTTCTTTCAGC-Tamra-3'; G3PDH forward: 5'-GAAGGTGAAGGTCGGAGT-3'; G3PDH reverse: 5'-CTTCTACCACTACCCTAAAG-3'; and G3PDH (Joe/Tamra) probe: 5'-Joe-CCGA- CTCTTGCCCTTCGAAC-Tamra-3'. Cycling parameters used were 2 min at 50°C, 30 s at 60°C, and 5 min at 95°C, followed by 40 cycles of 20 s at 94°C and 1 min at 60°C. The amount of mRNA in the sample was calculated from the number of the PCR cycle (threshold cycle; CT) at which an increase in reporter fluorescence above a baseline signal could first be detected. The CT is dependent on the starting template copy number (Heid et al., 1996). In this assay, CT values corresponding to the cycle number at which the fluorescent emission monitored in real time reached a threshold of 10 SD above the mean baseline emission from cycle 1 to cycle 15 were measured. The human iNOS mRNA expression was estimated by dividing the iNOS mRNA concentration by the G3PDH mRNA concentration.
Northern blot analysis of MMP-1 mRNA expression
Ten µg/lane of total RNA from CxF cells was separated on a 1.5% agarose gel containing 2% formaldehyde and transferred to Hybond-N+ nylon membranes (Amersham Pharmacia Biotech Ltd, Buckinghamshire, UK). Human proMMP-1 cDNA probe, a kind donation from Professor Hideaki Nagase (Kennedy Institute of Rheumatology, Imperial College, London, UK), was hybridized to the membranes using Rapid-Hyb Buffer (Amersham Pharmacia Biotech Ltd, Buckinghamshire, UK), followed by rinsing and final washes in 0.1xstandard saline citrate (SSC) and SDS for 5 min at 65°C. Densitometric analysis was performed using a BAS 2000 II Bioimage Analyzer (Fujix, Tokyo, Japan).
ELISA for MMP-1
The MMP-1 concentrations in the culture media from CxF cells were measured using a commercially available ELISA kit (Nippon Organon K.K., Osaka, Japan), which recognizes both proMMP-1 and the active mature form of MMP-1, as total MMP-1. The ELISA was conducted in duplicated wells and inter- and intra-assay variations of this system were 10% each.
Comparison between CxF cells established from pregnant women and those from non-pregnant women
In the current study, the responses of CxF cells from first trimester pregnant women (four cell lines) were similar to those from non-pregnant women (three cell lines), with respect to augmentation of iNOS expression by IL-1, increase in MMP-1 secretion in response to NO donors, and the blocking effect of L-NAME on IL-1-induced enhancement of MMP-1 secretion (data not shown). Based on these findings, we have only presented the data from experiments with the CxF cells established from pregnant women.
Statistical analysis
Values were expressed as mean ± SEM of quadruplicate dishes. Experiments were repeated three times and representative data are presented. The statistical significance was assessed by both the Mann–Whitney U-test and analysis of variance (ANOVA) followed by Fisher's protected least significant difference test. P < 0.05 was regarded as significant.
Results
Expressions of iNOS and eNOS in pregnant uterine cervical tissues and CxF cells
Strongly positive staining (brown) for iNOS was detected in cervical glandular cells and vascular endothelial cells of the uterine cervix from first trimester pregnant women (Figure 2A). Staining in the stromal cells was diffuse but clearly positive (Figure 2A). These results indicate the production of NO in uterine cervical tissue. Positive staining for eNOS was mainly detected in cervical glandular cells and vascular endothelial cells (Figure 2B). Negative controls using normal mouse IgG showed minimal staining (Figure 2C).
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RT–PCR analysis demonstrated iNOS mRNA expression in the uterine cervix from the first trimester of pregnancy (Figure 3A
; lanes 1,2) as well as in the CxF cells (Figure 3A; lanes 3,4). On the other hand, eNOS mRNA expression was detected in the uterine cervix in the first trimester of pregnancy (Figure 3B; lanes 1,2), but not in the CxF cells (Figure 3B; lanes 3,4). G3PDH mRNA expression is shown in Figure 3C as a control.
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Effect of IL-1
treatment on iNOS mRNA expression in CxF cellsQuantitative RT–PCR using TaqmanTM technology revealed that iNOS mRNA expression (iNOS mRNA/G3PDH mRNA) in CxF cells after 3 and 6 h of treatment with 1.0 ng/ml IL-1
was 0.16 ± 0.02 and 0.35 ± 0.09 respectively. Both of these values were significantly higher than those in vehicle controls (0.01 ± 0.01 each) (n = 3; P < 0.05 for both comparisons, Figure 4). However, iNOS mRNA expression (iNOS mRNA/G3PDH mRNA) in CxF cells after 12 h of treatment with IL-1 (0.03 ± 0.01) was similar to that in the vehicle control (Figure 4). Thus, IL-1, one of the key modulators of cervical ripening (Garcia-Velasco et al., 1999), was shown to augment iNOS mRNA expression in CxF cells.
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Effect of NO donors on MMP-1 production in CxF cells
The concentrations of MMP-1 in the culture media of CxF cells were measured by ELISA. After 24 h of treatment with SNP (10–6, 10–5 or 10–4 mol/l), the concentrations of MMP-1 were 13.7 ± 1.6 (n = 4), 14.0 ± 1.8, and 17.4 ± 4.3 ng/ml respectively, all of which were significantly higher than that of the vehicle control (8.5 ± 0.3 ng/ml) (P < 0.05 for all comparisons, Figure 5A
). This indicated a dose-dependent enhancement of MMP-1 by SNP. The MMP-1 concentrations in the culture media of CxF cells after 24 h of treatment with 10–5 and 10–4 mol/l DETA-NO were 15.5 ± 1.0 (n = 4) and 32.8 ± 3.3 ng/ml, both of which were significantly higher than that in vehicle control, 9.9 ± 3.3 (P < 0.05 for both comparisons, Figure 5B). This also indicated a dose-dependent enhancement of MMP-1 by DETA-NO. However, 8-bromo-cGMP, a membrane-permeable stable derivative of cGMP, did not alter MMP-1 secretion from CxF cells (Figure 5C). Northern blot analysis also revealed that treatment with SNP (10–4 mol/l) or DETA-NO (10–4 mol/l) augmented proMMP-1 mRNA expression in the CxF cells (Figure 6).
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Effects of a NO synthase inhibitor on IL-1
-augmented MMP-1 secretion from CxF cellsELISA measurement showed that IL-1
(1.0 ng/ml) could significantly (P < 0.05) augment MMP-1 secretion from cultured CxF cells. Treatment with L-NAME, an inhibitor of NOS, did not affect the basal secretion of MMP-1 from CxF cells (Figure 7). However, L-NAME partially, but significantly (P < 0.05), blocked the augmentation by IL-1 of MMP-1 secretion from CxF cells (Figure 7).
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After 24 h of incubation with various reagents used in the present study, the number of CxF cells was 2.5x105 to 2.8x105 cells/well in a 12-well plate. This was similar to that for the vehicle control.
Discussion
The present study demonstrated for the first time that the NO donors, SNP and DETA-NO, could augment MMP-1 mRNA expression in, and MMP-1 secretion from, cultured human uterine cervical fibroblast cells. A possible contribution of NO to the regulation of MMP-1 production has been previously reported for chondrocytes (Murrell et al., 1995; Lo et al., 1998). Since MMP-1 has a substrate selectivity for collagen type I and type III, the major components of the extracellular matrix of human uterine cervix, it is plausible that NO may directly stimulate MMP-1 production in uterine cervical fibroblast cells, thus promoting the degradation of extracellular collagens in the process of cervical ripening. In vascular smooth muscle cells, NO causes biological effects, including relaxation, by activating soluble guanylate cyclase, which generates cGMP as an intracellular second messenger (Garbers et al., 1994). However, 8-bromo-cGMP, a membrane-permeable stable derivative of cGMP, did not alter MMP-1 secretion from the cervical fibroblast cells. These data suggest the possibility that NO may augment MMP-1 secretion via a pathway distinct from soluble guanylate cyclase. Further studies of NO-associated signal transduction will be necessary to confirm this possibility.
iNOS expression was detected both in pregnant uterine cervix and cultured cervical fibroblast cells (Figures 2 and 3
). Furthermore, treatment with IL-1, one of the key initiators of cervical ripening (Garcia-Velasco and Arici, 1999), dramatically increased iNOS mRNA expression in cervical fibroblast cells (Figure 4), suggesting that IL-1 may up-regulate NO production in the uterine cervix at parturition. Consistent with this, IL-1 was also shown to enhance MMP-1 production from the cervical fibroblast cells. Moreover, treatment with L-NAME, an inhibitor of NOS, significantly and dose-dependently attenuated the IL-1-augmented MMP-1 secretion. However, treatment with L-NAME did not alter the basal secretion of MMP-1 from these cells. Since the concentration of IL-1 in cervical mucus is reported to increase greatly in pregnant women during labour (Cox et al., 1993), it is plausible that IL-1 may up-regulate MMP-1 secretion from uterine cervical fibroblast cells at least in part by stimulating local production of NO during parturition.
Moreover, all responses observed in cultured cervical fibroblast cells established from first trimester pregnant women were similar to those observed in cells from non-pregnant women. These findings suggest the possibility that pregnancy may not alter the regulatory mechanisms of the NO-associated increase of MMP-1 production in uterine cervix. Another possibility is that the characteristics of the original cervical tissues of non-pregnant and pregnant women were different, and that these differences were lost during culture in vitro. Another point to be considered is that the fibroblast cells used were obtained from pregnant women in the first trimester of pregnancy. Thus, the present study does not provide direct evidence of NO-mediated MMP-1 production in the uterine cervix at term.
To the best of our knowledge, this is the first report on the regulation of an MMP by NO as part of the complex cascade of cervical ripening. This provides a scientific basis for the recent report on the clinical application of NO donors as uterine cervical dilators (Thomson et al., 1998).
In summary, the present study demonstrated that iNOS is expressed in uterine cervix and cultured cervical fibroblast cells, and that treatment with IL-1 drastically increases the concentration of iNOS mRNA in the cervical fibroblast cells, suggesting a possible increase of NO production in the uterine cervix at parturition. Moreover, the study showed that treatment with NO donors enhances MMP-1 production in cervical fibroblast cells, and L-NAME partially, but significantly, blocks an augmentation of MMP-1 secretion by IL-1 from these cells. These data suggest a possible involvement of uterine cervical fibroblast NO production in the degradation of collagen during the process of cervical ripening.
Acknowledgements
The authors acknowledge Dr Masaki Harada MD, Dr Yoshihiko Saito MD, PhD, Professor Dr Kazuwa Nakao MD, PhD, Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, for their kind technical support of the study. The authors thank for Professor Hideaki Nagase, Kennedy Institute of Rheumatology Imperial College, UK, for generously donating the cDNA probe of proMMP-1. The authors also acknowledge Ms Akiko Kishimoto and Ms Akiko Abe for secretarial and technical assistance. This work was supported in part by Grants-In-Aid for Scientific Research from the Ministry of Education, Science, and Culture, Japan (No. 12877262, 13470352, 13557139, 13671707), a grant from the Ministry of Health and Welfare, and grants from the Smoking Research Foundation and the Kanzawa Medical Research Foundation.
Notes
4 To whom correspondence should be addressed. E-mail: fetus{at}kuhp.kyoto-u.ac.jp 
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Submitted on March 1, 2001; accepted on July 26, 2001.
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