1 Department of Medicine and
Research, Matrix metalloproteinases (MMPs) play an
important role in tumor metastasis and invasion, inflammatory tissue
destruction and remodeling, wound healing, and angiogenesis. The 72-kDa
gelatinase A is the most widely distributed of all the MMPs, and along
with the 92-kDa gelatinase B, both play an important role in the
turnover of basement membrane. The role of MMPs in chronic airway
inflammation and remodeling has received scant attention. In this
study, we sought to examine the release and activation of gelatinases
from human airway smooth muscle (ASM) cells and the effect of tumor necrosis factor-
human; matrix metalloproteinase; membrane-type matrix
metalloproteinase; tissue inhibitor of matrix metalloproteinase
MATRIX METALLOPROTEINASES (MMPs) play an important role
in tumor metastasis and invasion, inflammatory tissue remodeling and destruction, wound healing, and angiogenesis. The 72-kDa gelatinase A
(MMP-2) is the most widely distributed of all the MMPs (2). It is
expressed constitutively by most connective tissue cells including
endothelial cells, fibroblasts, myoblasts, osteoblasts, and
chondrocytes as well as invasive tumor cells. Along with the 92-kDa
gelatinase B (MMP-9), gelatinase A plays an important role in the
turnover of basement membrane type IV collagen (28) and in controlling
cell proliferation (24).
MMPs are tightly regulated in their transcription and release (15).
They are further regulated at the level of conversion of latent enzymes
to their active forms. Gelatinase A is secreted as a latent enzyme, and
when it is converted to a 62-kDa active form, it can degrade collagen
types IV and V, laminin, and elastin. The mechanism by which
progelatinase A is activated physiologically to a 62-kDa enzyme
involves a recently described, novel membrane-type matrix
metalloproteinase (MT-MMP) (23). Specific tissue inhibitors of
metalloproteinase (TIMPs) inhibit the activation of gelatinases. The
expression of TIMPs is also tightly regulated (18).
Chronic airway inflammation, reversible airway obstruction, and
bronchial hyperresponsiveness characterize bronchial asthma. This
airway inflammation involves microvascular leakage and release of
several inflammatory mediators including cytokines (3,
10). Pathologically, there is an eosinophilic and mast
cell infiltration of the submucosa, hyperplasia of mucous glands,
thickening of the subepithelial basement membrane, and hypertrophy and
hyperplasia of bronchial smooth muscle. This increase in airway smooth
muscle (ASM) mass may be as important as smooth muscle shortening in determining airway responsiveness in patients with asthma (10, 11, 26).
Recent observations have suggested that cytokines acting as mediators
of inflammation can stimulate vascular smooth muscle cells to increase
their production of gelatinase B (6). Gelatinases are necessary for
vascular smooth muscle proliferation (24) by virtue of their
degradation of vascular smooth muscle basement membrane, thereby
facilitating cell migration and subsequent cell proliferation. Other
investigators (29) have found that MMP inhibition decreased vascular
smooth muscle migration, proliferation, and intimal thickening in rat
arteries after injury. More recently, Ohno et al. (20) found gelatinase
B expression to be increased in eosinophils seen on bronchial biopsies
from asthmatic subjects.
In contrast to the active interest in vascular smooth muscle cells, the
effect of inflammatory mediators on ASM cell production and activation
of MMPs has received little attention. In this study, we sought to
systematically examine the release and activation of gelatinases from
ASM cells.
Materials
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
and phorbol 12-myristate 13-acetate
(PMA) on this release and activation. The role of membrane type 1 MMP
(MT1-MMP) and tissue inhibitor of MMP (TIMP)-2 in activating
progelatinase A has been explored. We have demonstrated, using human
airway smooth muscle cells in culture, that
1) ASM releases gelatinase A
constitutively and when stimulated with PMA and tumor necrosis factor-
releases gelatinase B, and the release of gelatinase B is
protein kinase C dependent because it is blocked by H-7 and staurosporin; 2) treatment of ASM
cells with PMA or concanavalin A failed to activate progelatinase A
despite these agents increasing cell expression of MT1-MMP; and
3) the inability of ASM cell
membranes to activate progelatinase A is most likely secondary to the
high levels of TIMP-2 on the cell membrane. In conclusion, our results demonstrate that human ASM cells constitutively secrete progelatinase A
and when stimulated with proinflammatory mediators secrete gelatinase B. The released gelatinases A and B may be important factors in the
airway remodeling that occurs in asthma.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
was purchased from Boehringer Mannheim
(Indianapolis, IN). Phorbol 12-myristate 13-acetate (PMA), aminophenylmercuric acetate, EDTA, H-7, staurosporin, concanavalin A
(Con A), and phenylmethylsulfonyl fluoride (PMSF) were purchased from
Sigma (St. Louis, MO). Precast zymogram gels were purchased from NOVEX
(San Diego, CA).
Recombinant human progelatinase A and recombinant (r) TIMP-1 and rTIMP-2 (19) were a generous gift from Celltech (Slough, UK).
Cell Culture
Human ASM Cells. Human tracheae were obtained from lung transplant donors in accordance with procedures approved by the University of Pennsylvania (Philadelphia, PA) Committee on Studies Involving Humans. ASM cells were cultured as previously described (22). Briefly, a segment of trachea just proximal to the carina was removed under sterile conditions, and the trachealis muscle was isolated. With this technique, ~0.5 g of wet tissue was obtained, minced, centrifuged, and resuspended in 10 ml of buffer containing 0.2 mM CaCl2, 640 U/ml of collagenase, 1 mg/ml of soybean trypsin inhibitor, and 10 U/ml of elastase. Enzymatic dissociation was performed for 90 min in a shaking water bath at 37°C. The cell suspension was filtered through 105-mm Nytex mesh, and the filtrate was washed with equal volumes of cold Ham's F-12 medium supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), 100 U/ml of penicillin, 0.1 mg/ml of streptomycin, and 2.5 µg/ml of amphotericin B. Aliquots of the cell suspension were plated at a density of 1.0 × 104 cells/cm2. The cells were refed with supplemented Ham's F-12 medium every 72 h. Cell counts were obtained with 0.5% trypsin in 1 mM EDTA. Human ASM cells in subculture during the second through the fifth cell passages were used because during these cell passages, the cells retain native contractile protein expression as demonstrated by immunocytochemical staining for smooth muscle actin and myosin (22).Human umbilical vein endothelial cells. Human umbilical vein endothelial cells (HUVECs) were harvested with the method of Jaffe (9) and cultured in gelatin-coated plates as previously described (5). The cells were grown in human endothelial-basal growth medium (GIBCO BRL, Life Technologies, Grand Island, NY) supplemented with 20% fetal bovine serum, 1% penicillin-streptomycin (GIBCO BRL), and 10 ng/ml of endothelial growth factor. All cells were grown in a humidified atmosphere of 95% air-5% CO2 at 37°C and passaged every 7-10 days. Cells from the third to the seventh passages were used.
Experimental Procedure
When ASM cells reached confluence in the wells, the cells were washed three times with serum-free medium. Test substances were added, and the cells were then incubated for 18 h in serum-free medium. Cell morphology (number of adhering cells, granularity, and spread) was examined after each incubation to substantiate that cell viability was not compromised by any of the test substances. The conditioned medium (CM) was then collected, centrifuged at 1,500 g for 10 min to remove any particulate matter and nonadherent cells, and then stored for 1-3 days atGelatinase Zymography and Reverse Zymography
Substrate zymography was performed in 10% polyacrylamide gels that had been cast in the presence of 0.1% gelatin. SDS-PAGE was performed with Tris-glycine SDS in sample and running buffers as described by the manufacturer (NOVEX). Samples were not boiled before electrophoresis. After electrophoresis, the gels were washed in 2.5% Triton X-100 to remove SDS, thus renaturing the gelatinases. The gels were then incubated in Tris buffer containing NaCl, CaCl2, and Brij 35 for 48 h at 37°C. Zones of enzymatic activity were characterized by the absence of Coomassie blue staining. Protein standards (Bio-Rad, Richmond, CA) were run concurrently, and approximate molecular masses of sample proteins were determined by plotting the relative mobilities of known proteins. Gelatinolytic bands were quantified by gel scanning and densitometry (30) with an Alpha Imager (Alpha Inotech, San Leandra, CA).For reverse zymography, we used gels impregnated with both 0.05% gelatin and activated gelatinase A (gelatinase A was aminophenylmercuric acetate activated). TIMP-2 bands were identified by Coomassie blue staining (8). Semiquantitative analysis of the amount of TIMP-2 was performed with gel scanning and densitometry.
Immunoblotting
To identify the secreted MMPs detected on the zymograms, immunoblotting of CM for gelatinase A was performed with affinity-purified specific rabbit antibodies to human gelatinase A as previously described (17).Immunoblotting of cell lysate for membrane type 1 MMP (MT1-MMP) was performed with affinity-purified rabbit antibodies directed against a unique peptide of MT1-MMP (CDGNFDTVAMLRGEM) (5). The cells were washed, then lysed by incubation in 1% SDS for 1 h. The samples were concentrated by precipitation in 6% TCA and resuspended in buffer. Prestained molecular-mass markers were run on each gel to identify molecular mass.
Immunoblotting of cell membranes for TIMP-2 was performed with affinity-purified rabbit antibodies directed against TIMP-2 supplied by Dr. Stetler-Stevenson (National Cancer Institute, Bethesda, MD) (30).
Quantification of Gelatinase A, Gelatinase B, TIMP-1, and TIMP-2
The concentrations of gelatinase A, gelatinase B (both latent and activated enzymes), TIMP-1, and TIMP-2 were determined in ASM CM with enzyme-linked immunosorbent assays (ELISAs) as previously described (12, 32). These experiments were repeated three times with samples run in duplicate.Preparation of Cell Membranes
Approximately 2 × 107 ASM or endothelial cells were rinsed three times in serum-free medium, mechanically scraped from dishes, pelleted by centrifugation, resuspended in cavitation buffer (25 mM sucrose and 5 mM MgCl2 in 5 mM Tris base, pH 7.4), and subjected to 1,000 psi N2 for 30 min at 4°C as previously described (33). Whole cells and nuclei were removed by centrifugation at 770 g for 10 min; the postnuclear supernatant was collected, and heavy organelles (mitochondria, lysosomes, and endoplasmic reticulum) were recovered by centrifugation at 6,000 g for 15 min. The supernatant was then centrifuged at 100,000 g for 1 h at 4°C to recover the lighter cell organelles in the pellet (i.e., plasma membranes, endoplasmic reticulum, and Golgi); the 100,000-g supernatant was collected and designated cytosol.Cell Surface Binding of TIMP-2
To examine TIMP-2 binding to ASM and compare it with that of TIMP-2 binding to HUVECs, we used methods previously described (31). Briefly, rTIMP-2 was iodinated to a specific activity of 5.5 ×1010 dpm/mg by adding 0.25 mCi of Na125I to a tube containing 10 µg of rTIMP-2 and 100 µg of chloramine T. After a 5-min incubation on ice, 200 µg of sodium metabisulfite were added, and 125I-labeled TIMP-2 in PBS containing 0.1% BSA was separated from free 125I by chromatography over a G-25 Sephadex column (Pharmacia Biotech, Washington, DC). Binding of 125I-labeled TIMP-2 to cells propagated in 24-well dishes (Corning Costar, Wilmington, MA) to >90% confluence was performed in duplicate ( ![]() |
RESULTS |
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Release and Activation of Progelatinases A and B
Gelatin zymography of CM from untreated ASM showed an incremental increase over time in the release of 72-kDa gelatinolytic bands, consistent with progelatinase A (data not shown). To confirm the identification of the gelatinolytic bands released by ASM, progelatinase A (72 kDa) was identified on immunoblots with specific rabbit anti-human antibodies to gelatinase A (data not shown).PMA and the tetravalent lectin Con A are known to induce several cell
types to activate progelatinase A (5, 21, 27). When ASM cells were
treated with PMA, the zymogram of the CM showed an increase in
gelatinolytic activity at 92 kDa (Fig.
1A)
and a variable increase at 85 kDa (best visualized in Fig.
1B), consistent with progelatinase B
and activated gelatinase B, respectively; however, there was no change
in the gelatinolytic activity at 72 kDa (progelatinase A) and no
activation. PMA also induced gelatinolytic activity at ~55 kDa,
presumably representing collagenase or stromelysin-1 (Fig.
1A). Treatment of ASM cells with
Con A likewise did not result in activation of progelatinase A (see
Fig. 5).
|
Based on the known release of numerous cytokines during inflammation,
we examined the effect of TNF- on ASM progelatinase A activation.
ASM cells were treated with TNF-
(103 to
105 U) for 24 and 48 h. TNF-
induced a small increase in the 92- and 85-kDa gelatinolytic bands
(progelatinase B and activated gelatinase B, respectively; best
visualized in Fig. 1B) but had little effect on progelatinase A in the CM. When TNF-
was added in
combination with PMA to ASM cells, the CM exhibited a large increase in
the prominence of the latent and activated gelatinase B bands at 95 and
85 kDa, respectively. This increase in progelatinase B with TNF-
and
PMA was dose dependent (Fig. 1A)
and mediated through protein kinase (PK) C because it was inhibited by
the PKC inhibitor H-7 (Fig. 1B) and
staurosporin (data not shown). These results were confirmed by an ELISA
that demonstrated a large increase in immunoreactive gelatinase B in
the CM of cells treated with the combination of PMA and TNF-
(data
not shown) but no change in measured gelatinase A.
We then compared CM from ASM cells and HUVECs using an ELISA to measure
gelatinase A. The levels of gelatinase A in the CM from ASM cells and
HUVECs were similar (3.32 ± 1.02 and 3.98 ± 0.44 nM,
respectively; Table 1).
|
TIMP-1 and TIMP-2 Release From ASM Cells and HUVECs
CM from ASM cells and HUVECs was analyzed with ELISAs to measure TIMP-1 and TIMP-2 levels. We found that ASM cells secreted considerably larger amounts of TIMP-1 compared with HUVECs (323.49 ± 29.3 and 1.31 ± 0.8 nM, respectively). However, TIMP-2 in the CM of ASM cells was lower than that in HUVECs (0.35 ± 0.04 and 0.96 ± 0.09 nM, respectively; Table 1).Mechanisms of ASM Progelatinase A Activation
In contrast with ASM cells, PMA and Con A treatment of HUVECs results in activation of secreted progelatinase A (5). Cell membranes isolated from HUVECs readily activated progelatinase A in HUVEC CM (13), thus implicating a plasma membrane activator, presumably MT1-MMP (23).To investigate the inability of PMA or Con A to activate ASM
progelatinase A while being effective in activating progelatinase A
from HUVECs, cell membrane fractions from HUVECs were incubated for 18 h with CM from ASM cells. The addition of the plasma membrane-enriched fraction (100,000-g pellet) from
HUVECs to the ASM CM resulted in a dose-dependent increase in
gelatinolytic bands at 64 and 62 kDa, which is consistent with the
activation of progelatinase A (Fig.
2A).
When the experiment was altered so that cell membranes from ASM cells
were incubated with CM from ASM, no activation of progelatinase A was
noted (Fig. 2B).
|
To examine whether HUVEC membrane activation of gelatinase A was
specific, we added HUVEC plasma membranes to CM from ASM in the
presence and absence of TIMP-2, TIMP-1, PMSF (serine proteinase inhibitor), and E-64 (cysteine proteinase inhibitor). We found that
TIMP-2 inhibited the HUVEC membrane activation of progelatinase A, but
TIMP-1 did not (Fig. 3). PMSF and E64 were
also unable to prevent this activation (data not shown). These results
suggest that the plasma membranes of HUVECs, but not of ASM, contain an essential component required for the activation of progelatinase A.
|
To determine whether culture conditions affected progelatinase A activation, ASM cells were cultured on a gelatin substrate. This had no effect on gelatinase A release or activation (data not shown).
A recent study (23) has suggested that MT1-MMP, an intrinsic membrane
protein, is responsible for cell-mediated activation of progelatinase
A. Based on this information, cell membrane preparations from ASM cells
and HUVECs were examined by immunoblotting for the presence of MT1-MMP.
Our immunoblot results suggest that ASM and HUVEC plasma membranes
contain approximately equivalent amounts of MT1-MMP (Fig.
4). Northern blot analysis revealed an
upregulation of MT1-MMP mRNA in ASM cells treated with PMA or Con A
(Fig. 5). In HUVECs, PMA caused an increase
in MT1-MMP mRNA, whereas Con A did not (data not shown).
|
|
When membrane preparations from ASM were combined with HUVEC membrane
preparations (100,000-g pellet), the
ability of HUVEC membranes to activate progelatinase A in ASM CM was
diminished; this result suggests the presence of an inhibitor of
progelatinase A activation in the membranes of ASM cells. The
inhibitory effect of ASM membranes on HUVECs was diminished with
further dilution of ASM membranes (Fig. 6).
|
To further investigate the differences between crude plasma membrane
preparations from HUVECs and ASM cells, we examined gelatinase A
immunoblots. In HUVEC membranes, gelatinase A was identified primarily
as a 64-kDa band, with a weaker dimer at ~72 kDa, whereas in ASM
membranes, gelatinase A was present primarily as a 72-kDa band (Fig.
7). These data are consistent with
activation of progelatinase A after binding to HUVEC plasma membrane.
In ASM, progelatinase A binding to plasma membranes was minimal and no
enzyme activation was noted.
|
TIMP-2 in ASM and HUVEC Membranes
Using immunoblotting and reverse zymography, we identified TIMP-2 bands on ASM and HUVEC crude membranes. Our densitometry results show that seven to nine times more TIMP-2 (21 kDa) was associated with ASM than with HUVEC membranes (Fig. 8). Reverse zymography also identified a 50-kDa band in both ASM and HUVEC membranes that has been previously reported (16). In the TIMP-2 Western blot of ASM membranes, there were several bands between 30 and 42 kDa. These bands have been previously described in other biological samples (17) and are of unknown significance. To determine whether the bands identified on reverse zymography were TIMP-1, we performed Western blots on CM and membranes from ASM and HUVECs. TIMP-1 was readily detected in the CM from ASM cells but not in the CM from HUVECs; TIMP-1 was not detected in the cell membrane of either cell type (Fig. 9).
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125I-Labeled TIMP-2 Binding to ASM Cells and HUVECs
125I-labeled TIMP-2 binding to PMA-treated ASM cells demonstrated a specific and saturable pattern of binding, with a dissociation constant (Kd) of 5.37 nM and a maximum binding capacity (Bmax) of 0.048 nM (Fig. 10, A and B). The number of receptors per ASM cell was
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DISCUSSION |
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MMPs are important in inflammatory tissue destruction and remodeling.
In this study, we systematically investigated the release and
activation of gelatinases A and B from ASM cells and further used these
cells to study the mechanism of progelatinase A activation. Our results
demonstrate that ASM cells secrete progelatinase A constitutively and
when stimulated with PMA and/or TNF-, these ASM cells were induced
to release gelatinase B. The release of gelatinase B through a
PKC-dependent mechanism is similar to gelatinase B regulation in aortic
smooth muscle (4). CM from ASM cells had large amounts of TIMP-1 and a
relatively small amount of TIMP-2 compared with those in HUVECs.
The mechanism of cell surface-mediated activation of progelatinase A is of considerable current interest. Unlike what Foda et al. (5) and others (7) have reported with HUVECs, ASM progelatinase A is not activated by cells treated with PMA or Con A. We took advantage of this difference to elucidate the activation of progelatinase A. PMA- and Con A-induced HUVEC progelatinase A activation occurs through a plasma membrane-dependent mechanism (5). Progelatinase A in ASM CM was readily activated when incubated with HUVEC membrane preparations but was not activated when incubated with ASM membranes, suggesting a lack of an important "inducing factor" or the presence of an inhibitor in the membranes of ASM. The activation of progelatinase A by HUVEC membranes was specific because it was inhibited by the addition of excess TIMP-2 but not by TIMP-1. Furthermore, it is unlikely that the initial progelatinase A activation step (72-64 kDa) induced by HUVEC membranes was due to autoactivation because it was not inhibited by excess TIMP-1, which inhibits autoactivation of soluble progelatinase A (30). The data in Fig. 6, indicating that small amounts of ASM membrane have an inhibitory effect on HUVEC membrane-induced progelatinase A activation, is consistent with the presence of an inhibitory factor in ASM membranes.
Strongin et al. (25) and Zucker et al. (31) have proposed that the plasma membrane-dependent activation of progelatinase A on HT 1080 fibrosarcoma cells and endothelial cells required the formation of a trimolecular complex of progelatinase A, TIMP-2, and MT1-MMP on the cell surface (23). Free MT1-MMP (not complexed with TIMP-2) is required to convert 72-kDa progelatinase to a 64-kDa intermediate (31), which is followed by autoactivation of 64- to 62-kDa-activated gelatinase A (1, 25). We demonstrated 64-kDa gelatinase A on crude plasma membranes from HUVECs but not from ASM, which suggests both lack of binding and activation of progelatinase A on ASM. Our results indicate that although there is increased mRNA expression of MT1-MMP in ASM cells, activation of progelatinase A was not induced. In contrast, the addition of Con A to HUVECs leads to the activation of progelatinase A (5) but no increase in MT1-MMP mRNA.
Cell membranes of ASM contain a considerable amount of TIMP-2 that we suspect totally saturates the MT1-MMP binding capacity for TIMP-2. Therefore, no free MT1-MMP is available to activate gelatinase A bound to the triplex of MT-MMP1, TIMP-2, and gelatinase A. We therefore conclude that the excess TIMP-2 in the membrane contributes to the inhibition of gelatinase A activation in ASM. The relatively low levels of TIMP-2 in ASM CM is presumably secondary to high amounts of TIMP-2 bound to ASM membranes. In the experimental conditions employed for evaluating 125I-TIMP-2 binding, the cell surface is stripped of bound TIMP-2 by lowering the pH, thus permitting maximum binding of radiolabeled ligand. This effect was exaggerated with ASM compared with that with HUVECs (data not shown) and is consistent with excess endogenous TIMP-2 bound to ASM membranes. Additional studies are needed to explain the mechanism for intense binding of endogenous TIMP-2 to ASM membranes even though TIMP-2 levels are higher in HUVEC CM than in ASM CM. An abnormality of MT1-MMP function on insertion in the plasma membrane is suspected; a higher Kd for 125I-TIMP-2 binding to ASM cells compared with that to HUVECs supports this assumption.
Lohi et al. (14) have recently reported findings similar to ours in embryonic lung fibroblasts where PMA did not activate progelatinase A despite an increase in MT1-MMP mRNA. The authors suggested that this was due to the lack of a "cooperating factor"; however, they did not consider the possibility of an inhibitory phenomenon.
In conclusion, our results demonstrate that human ASM cells constitutively secrete progelatinase A and when stimulated with proinflammatory mediators secrete gelatinase B. The released gelatinases A and B may be important factors in the airway remodeling that occurs in asthma.
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ACKNOWLEDGEMENTS |
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We thank Dr. William G. Stetler-Stevenson (National Cancer Institute, Bethesda, MD) and Dr. Andrew J. P. Docherty (Celltech Ltd., Slough, UK) for their generous gifts of material.
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FOOTNOTES |
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This work was supported by funds from the American Lung Association of New York and the American Heart Association (to H. D. Foda) and by Veterans Affairs Merit Review Grant Funds (to S. Zucker).
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: H. D. Foda, Pulmonary and Critical Care Medicine, SUNY at Stony Brook, Health Science Center, Stony Brook, NY 11794-8172 (E-mail: hfoda{at}mail.som.sunysb.edu).
Received 31 August 1998; accepted in final form 10 March 1999.
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