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Original Articles: Hormones and Reproductive Health |
Departments of Gynecology and Obstetrics (R.M.P., M.L., G.H.F., L.C.G.), Pediatrics (S.B.), and Radiation Oncology (A.J.G.), Stanford University Medical School, Stanford, California 94305; and Department of Gynecological Endocrinology and Reproductive Medicine (R.M.P.), Ruprecht-Karls University, Heidelberg, Germany
Address all correspondence and requests for reprints to: Linda C. Giudice, Ph.D., M.D., Division of Reproductive Endocrinology and Infertility Center for Research on Women’s Health and Reproductive Medicine, Department Gynecology and Obstetrics, Stanford University Medical Center, Room HH-333, Stanford, California 94305-5317. E-mail: giudice{at}stanford.edu
Abstract
Intrauterine growth restriction (IUGR) can be a consequence of decreased uterine blood flow (uteroplacental insufficiency) and maternal and fetal hypoxia. Insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) are key elements in fetal growth. IGF-I is a major growth promoter in utero. IGFBP-1 is primarily made in the liver, and it mostly inhibits IGF actions at the cellular level. IGFBP-1 is elevated in the fetal circulation of human and animal pregnancies complicated by IUGR caused by placental insufficiency and in utero hypoxia and is believed to restrict fetal growth by sequestering IGFs. In this study, we developed a protocol to establish highly pure primary cultures of human fetal hepatocytes in vitro and investigated their expression of IGFBP-1 messenger RNA (mRNA) and protein and the effects of hypoxia on their expression of IGFBP-1 mRNA and protein. Hepatocytes were isolated from second-trimester human fetal livers (n = 7) and purified by Percoll gradient centrifugation. Hepatocyte cultures were characterized by immunocytochemistry and were compared with hepatocytes in situ in human fetal liver tissue, by immunohistochemistry, using specific antibodies and indirect immunofluorescence. Cultures consisted primarily (>90%) of cells positive for cytokeratin 18, fibrinogen, and IGFBP-1, with less than 2% vascular cells and less than 8% macrophages. Identification of isolated hepatocytes was further confirmed by morphology. Hepatocytes were cultured in defined medium, and Northern analysis revealed expression of a 1.5-kb IGFBP-1 mRNA transcript in hepatocytes cultured under normoxic conditions, for 24 h, that did not increase in steady-state levels after 48 h in culture. Under hypoxic conditions (2% O2), IGFBP-1 mRNA expression increased 3- to 4-fold, compared with normoxic controls. Cells cultured under 10% O2 did not demonstrate an increase in IGFBP-1 mRNA levels. IGFBP-1 protein in conditioned medium (CM) was measured by immunoradiometric assay and increased 3- to 4-fold under hypoxic (2% O2), compared with normoxic, conditions. Western ligand blot analysis of CM revealed the presence of IGFBP-1, IGFBP-2, IGFBP-3, and IGFBP-4. IGFBP-1 was the most abundant IGFBP in CM, and densitometric analysis revealed a 2.5-fold increase in IGFBP-1 under hypoxic, compared with normoxic, conditions, supporting the immunoradiometric assay results. A 3-fold increase in IGFBP-3 mRNA, but not other IGFBPs, was noted under hypoxic, compared with normoxic, conditions. This study demonstrates that human fetal hepatocytes can be cultured in defined medium, as primary cultures with high purity, and that they express IGFBP-1 mRNA and secrete IGFBP-1 protein in vitro. In addition, the data demonstrate that hypoxia up-regulates fetal hepatocyte IGFBP-1 mRNA steady-state levels and protein, with this being the major IGFBP derived from the fetal hepatocyte. The data support a role for the fetal liver as a source of elevated circulating levels of IGFBP-1 in fetuses with in utero hypoxia and IUGR.
FETAL SIZE AND weight are regulated by complex mechanisms, including genetic and environmental factors (1). Morbidity and mortality are greatly increased in fetuses with intrauterine growth restriction (IUGR) (2). The most common cause of IUGR is placental insufficiency characterized by decreased nutrients and O2 supplied to the placenta and, therefore, to the fetus. Members of the insulin-like growth factor (IGF) family, including the growth-promoting IGF peptides and IGF-binding protein (IGFBP)-1, an IGF inhibitor, have been implicated in normal and abnormal fetal growth, in humans as well as in animal models (3, 4). For example, IGF-I and IGF-II null mice are born severely growth restricted (5, 6), and IGFBP-1 transgenic mice have approximately 15% lower birthweights, compared with their wild-type littermates (7). A partial deletion in the coding region of the IGF-I gene results in severe prenatal and postnatal growth in humans (8). Also, clinical studies in humans demonstrate direct correlations between IGF-I in fetal cord blood or serum and fetal size and birthweight (9, 10, 11, 12, 13), and a striking inverse correlation exists between fetal and maternal IGFBP-1 levels and birthweight (9, 11, 14, 15, 16, 17, 18). Cordocentesis at 26–27 weeks demonstrates an increase of more than 2-fold in IGFBP-1 and an IGF-I level 4-fold lower in fetuses with uteroplacental insufficiency (UPI) and IUGR, compared with normal weight, control fetuses (19).
Immunohistochemical studies (20) and studies with human fetal tissue explant cultures (21, 22) demonstrate that IGFBP-1 is primarily expressed in fetal liver, with lower amounts present in fetal kidney, suggesting that the liver is the major source of circulating IGFBP-1 in the fetus, as in adults (23, 24). The stimulus for elevated IGFBP-1 in the fetal circulation in pregnancies complicated by IUGR has not been determined. There are multiple hormones implicated in fetal growth and development that have regulatory elements in the promoter region of the IGFBP-1 gene (25). Glucocorticoids, associated with fetal stress, have been suggested to elevate IGFBP-1 levels in IUGR fetuses caused by placental insufficiency (26, 27). Alternatively low levels of circulating insulin, which is a potent inhibitor of IGFBP-1 (23, 24), may contribute to elevated IGFBP-1 levels. The finding of a functional consensus sequence for the hypoxia response element in intron 1 of the IGFBP-1 gene (28) and hypoxic induction of IGFBP-1 gene expression in HepG2 cells (28), as well as the association of intrauterine hypoxia and elevated IGFBP-1 levels in human fetuses (28), all suggest that hypoxia may regulate IGFBP-1 in human fetal liver. Supporting this hypothesis are animal models of maternal hypoxia and/or intrauterine hypoxia and nutrient restriction caused by uterine artery ligation, in which markedly elevated IGFBP-1 messenger RNA (mRNA) levels are found in the fetal liver with concomitant elevation of IGFBP-1 in the fetal circulation (12, 18, 29, 30, 31). The goals of the current study were: 1) to isolate and culture human fetal hepatocytes in defined medium and with high purity; 2) to investigate whether cultured human fetal hepatocytes express IGFBP-1 mRNA and secrete IGFBP-1; and 3) to investigate whether cultured human fetal hepatocytes increase IGFBP-1 production in response to hypoxia. The data support a role for hypoxia in the induction of IGFBP-1 observed in the human fetus under conditions of UPI and in utero hypoxia. By sequestering IGFs, IGFBP-1 may inhibit IGF-mediated fetal growth, thereby contributing to IUGR that is commonly found with UPI and in utero hypoxia.
Materials and Methods
Samples
Fetal liver tissue was obtained after informed consent at the time of elective terminations of pregnancy of second-trimester, chromosomally normal fetuses from uncomplicated pregnancies at Stanford University Hospital (Stanford, CA). The protocol was approved by the Stanford University Committee in the Use of Human Subjects in Medical Research. Liver samples (n = 7; 4 male, 3 female), ranging from 18–22 weeks of gestation, were immediately collected in DMEM (Life Technologies, Inc., Grand Island, NY).
Cell isolation and cell culture
Samples were washed three times with sterile PBS before removing the gallbladder and all visible vessel branches. The remaining liver tissue was then minced with forceps and a scalpel and treated with liver digest medium (Life Technologies, Inc.) containing 0.1% collagenase/dispase per gram wet weight of tissue, at 37 C for 1 h with gentle agitation. Liver cells were collected by gradient centrifugation (3 x 5 min at 500 rpm) to separate them from endothelial and biliary tract cells and purified of red blood cells over a 50% Percoll gradient. Hepatocytes were collected and counted, and their viability was determined to be approximately 96% using trypan blue exclusion. Cells were resuspended in serum-free Hepatocyte Attachment Medium (Life Technologies, Inc.) supplemented with penicillin/streptomycin and 5 µg/mL bovine fibronectin (Roche Molecular Biochemicals, Indianapolis, IN) and were plated at a density of 10 x 106 cells/20 cm2 permanox plastic plates, which had been previously coated with type IV collagen (Sigma, St. Louis, MO). After overnight attachment, unattached cells were removed, and medium was changed to serum-free Hepato Zyme Serum Free Medium (Life Technologies, Inc.), supplemented with L-glutamine and antibiotics. At this plating density, attached hepatocytes were about 60% confluent at the beginning of each experiment. Experiments were always performed the same day (1 day after sample collection).
Characterization of cell culture by immunocytochemistry (ICC)
Isolation and culturing fetal hepatocytes in primary culture have not, to our knowledge, been previously described. Therefore, characterization of hepatocyte cultures was conducted by ICC, and comparison was made with hepatocytes in situ in human fetal liver tissue, by immunohistochemistry (IHC), using an indirect immunofluorescent technique. Identity of cultured hepatocytes was further confirmed by their typical morphology. For immunohistochemical analyses, human fetal liver tissues (n = 4) were embedded in cold OCT compound (Tissue Tek, Elkhart, IN) and frozen in dry ice. Cryostat sections, 4-µm thick, were mounted on slides and fixed with 100% acetone (histograde) for 10 min and were air-dried. For ICC, hepatocytes were isolated and cultured on permanox slides coated with collagen type IV (Sigma), and they were processed as with the cryostat sections. Samples were incubated with the following primary antibodies: mouse antihuman cytokeratin 18 (1:50) (Sigma), rabbit antihuman fibrinogen (1:200) (DAKO Corp., Carpinteria, CA), mouse antihuman IGFBP-1 monoclonal antibody (1:100) (Diagnostic Systems Laboratories, Inc., Webster, TX); mouse antihuman CD 68 (1:100) (DAKO Corp.), and rabbit antihuman Factor VIII (1:100) (Sigma). Negative controls were incubated with mouse ascites fluid and rabbit IgG for antibodies derived from mouse and rabbit, respectively. After washing three times with PBS and 0.1% BSA, sections and cells were incubated with a secondary antibody, fluorescein isothiocyanate-conjugated or Texas red antimouse IgG or antirabbit IgG (Zymed Laboratories, Inc., San Francisco, CA), for 60 min at 25 C. Some slides were also counterstained with DAPI (4', 6-diamidino-2-phenylindole, Vectrashield Products, Burlingame, CA) for localization of nuclei. Slides were photographed using a Carl Zeiss 35-mm camera and a Carl Zeiss microscope (MC80 DC, Thornwood, NY), fitted with a BH-RFL-W reflected light fluorescence attachment, using three filters for red, green, and blue fluorescence.
Hypoxia treatment
Medium was changed freshly before the start of each experiment. To determine the effect of hypoxia on IGFBP-1 production of fetal hepatocytes, all cells were placed in specially designed aluminum hypoxia chambers, which were preheated at 37 C, sealed, and subjected to successive rounds of evacuation, followed by flushing with 95% N2O-5% CO2 mixture to produce a specific pO2 as determined by a Clark-type O2 electrode (Controls Katharobic, Edmonton, Canada) (32, 33) for 24 and 48 h. After one cycle of evacuation, the O2 concentration in the media was reduced to 2%. The aluminum chambers were then placed into an incubator at 37 C for up to 72 h. Controls were cultured in normoxia (20% O2) for 24 and 48 h. Conditioned media were collected, centrifuged, and stored for further analysis at -80 C. Total RNA was isolated as described below.
Northern blot analyses
Northern blotting was performed using the method of Chomczynski
and Sacchi (34) and modified as previously described
(35). Briefly, RNA was isolated from hepatocytes using
Trizol (Life Technologies, Inc., Carlsbad, CA),
according to the manufacturer’s instructions. Eight micrograms of RNA
were heated and denatured in a formamide/formaldehyde buffer containing
ethidium bromide (Sigma), and electrophoresis was
conducted on 1.2% agarose-formaldehyde gel. RNA was assessed for
integrity and equivalency in loading by ultraviolet visualization of
the ethidium bromide stain and was transferred to nitrocellulose
membrane (Schleicher & Schuell, Inc., Keene, NH) by
capillary transfer. Membranes were then probed using a 938-bp
EcoRI fragment of the human IGFBP-1 complementary DNA
(36). The probe was labeled with deoxycytidine
[
-32P] deoxycycidine triphosphate (NEN Life Science Products, Boston, MA) using a random primer
kit (Ready-to-Go, Pharmacia Biotech, Piscataway,
NJ). Membranes were blocked with Express Hyb (CLONTECH Laboratories, Inc. Palo Alto, CA) at 68 C and probed for 1
h using 1 x 106 cpm/mL of the random-primed
probe at 68 C. Membranes were washed three times at 25 C with 2x SSC
and 0.05% SDS and twice at 55 C with 0.1x SSC and 0.1% SDS.
Autoradiography was performed at -70 C for 12 h to 4 days. The
molecular size was determined using an RNA ladder (Promega Corp., Madison, WI).
Densitometry
Autoradiographs of bands on Northern blots and of ethidium bromide staining of 18S RNA were analyzed on a PDI Desk Top Scanner. The integrated areas under the absorbance curves were measured for each band and were used to determine the relative amounts of 18S mRNA and IGFBP-1 mRNA.
Western ligand blot analysis
IGFBPs in conditioned medium (CM) of human hepatocytes were analyzed by ligand blotting, according to the method of Hossenlopp et al. (37), as described previously (11). Briefly, nonreduced samples of CM (90 µL) were electrophoresed on 12% SDS acrylamide gels under nonreducing conditions and electrotransferred onto nitrocellulose. The blotted proteins were incubated with 1,000,000 cpm 125I IGF-I overnight at 4 C, washed, air-dried, and exposed to radiographic film.
Immunoradiometric assay
Conditioned medium was assayed for total IGFBP-1 by immunoradiometric assay (Diagnostics Systems Laboratories, Inc.), according to manufacturer’s instructions. The inter- and intraassay coefficients of variance were 8.5% and 6.5%, respectively. IGFBP-1 standards were plotted as a linear function.
Statistical analysis
All experimental variables were tested in duplicate cultures in each experiment. Specific IGFBP-1 densitometry values, normalized to 18S, were used to calculate mean values ± SEM and fold increase of IGFBP-1 signals relative to time-matched controls. Levels of IGFBP-1 protein in CM of duplicate plates were used to calculate mean values ± SEM and fold increase of treatment groups, compared with time-matched controls. Data were analyzed by ANOVA using the Stat-View software (Abacus Concepts, Berkeley, CA). Significance between treatment group means was determined using Fisher’s protected least-significant difference post hoc test, with P < 0.05 taken as significant.
Results
Human fetal hepatocyte cultures
The characterization of primary hepatocyte cultures was conducted
by ICC, and comparison was made to immunostaining of hepatocytes
in situ in human fetal liver tissue, by IHC, using an
indirect immunofluorescent technique, as described in Materials
and Methods. Figure 1
depicts the
IHC results (left panel) and the ICC results (right
panel). The cells in lane 1 were immunostained with the stated
antibody or antiserum. The cells in lane 2 were counterstained with
DAPI to show the presence of all cell types present in the tissue and
cultures, and the cells in lane 3 demonstrate nonimmune staining. Rows
A–C depict examples of specific immunostaining for cytokeratin 18
filaments, fibrinogen in the cytoplasm, and IGFBP-1 in the cytoplasm,
respectively. These IHC markers are characteristic of liver epithelial
cells (cytokeratin 18) and specific fetal hepatocyte products
(fibrinogen and IGFBP-1). Row D illustrates vascular endothelial cell
staining with anti-Factor VIII antibody, and row E demonstrates
specific staining for macrophages with anti-CD 68 antibody. The
immunocytochemical studies (right panel) reveal that the
hepatocyte cultures consisted predominantly of cytokeratin 18-positive,
fibrinogen-positive, and IGFBP-1-positive cells (>90%), with less
than 2% vascular endothelial cells and less than 8% macrophages.
Identity of cultured hepatocytes was further confirmed by their
morphology.
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Primary cultures of human fetal hepatocytes were established, and
the effect of hypoxia over a 48-h period on their production of IGFBP-1
mRNA and protein was investigated (Fig. 2). Under normoxic conditions, Northern
analysis revealed that human fetal hepatocytes expressed a 1.5-kb mRNA
encoding IGFBP-1. Under hypoxic conditions (2%
O2), there was a marked increase in the intensity
of the 1.5-kb mRNA signal (Fig. 2A). Densitometric analysis of Northern
blots from five different experiments (i.e. five different
liver samples), in duplicate, revealed a 3.0 ± 0.91-fold increase
of IGFBP-1 mRNA at 24 h, which did not increase significantly with
further exposure to hypoxia (48 h) (Fig. 2B). We also investigated the
induction of IGFBP-1 at 10% O2. Under these
conditions, IGFBP-1 expression was identical to that of cells exposed
to 20% O2 (Fig. 3), whereas the steady-state levels of
IGFBP-1 mRNA in hepatocytes cultured in 2% O2
was approximately 3-fold higher than under normoxic conditions.
Exposure of hepatocytes to hypoxic conditions of 0.2% and 0.02%
O2 revealed detectable, but decreased, IGFBP-1
mRNA expression (data not shown), presumably caused by decreased cell
viability. Under normoxic conditions, IGFBP-1 protein levels in medium
conditioned by primary cultures of human fetal hepatocytes (Fig. 2C)
were 13 ± 5 ng/mL·106 cells at 24 h
of culture. Under hypoxic conditions (2% O2),
IGFBP-1 protein levels increased to 35.9 ± 9
ng/mL·106 cells (P < 0.05). At
48 h of normoxic exposure, the levels of IGFBP-1 protein in
the medium were 35.5 ± 13 ng/mL·106
cells; and under hypoxic conditions, they rose to 70.9 ± 10
ng/mL·106 cells (P < 0.05).
Overall, there was a 2.5 ± 0.3-fold increase in IGFBP-1 in CM
under hypoxia vs. normoxia, which was in good agreement with
the increase in steady-state levels of IGFBP-1 mRNA.
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shows an autoradiograph of conditioned
media from hepatocytes isolated from two different liver samples
cultured for 24 h and 48 h under hypoxic or normoxic
conditions. Under both conditions, a major band of 28 kDa, which
represents IGFBP-1 (22), was observed. We also examined
the effects of hypoxia on the IGFBP-3 doublet (38–43 kDa), IGFBP-2
(migrating at 32 kDa), and IGFBP-4 (24 kDa). Densitometric analysis of
the autoradiogram shown in Fig. 4A is presented in Fig. 4B. A 2-fold
increase of IGFBP-1 (28-kDa band) was seen in the CM of hepatocytes
maintained in 2% O2, both at 24 h and at
48 h, consistent with the quantitative analysis performed on
IGFBP-1 in CM, presented in Fig. 2C
. Hypoxia also caused a significant
increase of IGFBP-3 that reached a maximum of 3-fold (P
< 0.05), whereas IGFBP-2 and IGFBP-4 increased only slightly under
hypoxic conditions, and these increases were not statistically
significant (Fig. 4B).
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In this report, we have presented a method to isolate second-trimester human fetal hepatocytes and culture them with high purity in defined medium. We have demonstrated that human fetal hepatocytes in culture express IGFBP-1 and respond to hypoxia by increasing IGFBP-1 mRNA steady-state levels and secreted protein levels. Traditionally, human hepatocellular carcinoma cell lines have provided useful models to investigate hepatic gene regulation by hypoxia (28). Although these cell lines are valuable model systems to study gene regulation or when fetal hepatocytes are unavailable, they may not be reflective of human fetal hepatocyte physiology. The human fetal hepatocyte culture system reported here provides a useful model to investigate human fetal hepatocyte physiological responses to changes in O2 tension and to investigate experimental paradigms that cannot be addressed in the human fetus in vivo.
The IGF family (and in particular, IGF-I and IGFBP-1) are important in fetal growth. In humans, IGF-I levels are decreased and IGFBP-1 levels are increased up to 20-fold in the fetal circulation under conditions of UPI and IUGR (9, 11, 14, 15, 16, 17). Babies with severe IUGR and UPI are hypoxic and hypoinsulinemic and have elevated levels of IGFBP-1 and glucocorticoids, as well as counterregulatory hormones, such as glucagon (2). Our observations that human fetal hepatocytes increase production of IGFBP-1 mRNA and protein in response to hypoxia support the hypothesis that, under conditions of in utero hypoxia, fetal hepatocytes have the capacity to increase IGFBP-1 production and may be the primary source of elevated circulating IGFBP-1. Of note is zonal expression of IGFBP-1 mRNA in adult rat liver (38), with differential expression of IGFBP-1 in the perivenous vs. periportal zones, believed to be regulated by O2 tension and metabolic factors. It is likely that the liver is a major contributor to elevated circulating levels of IGFBP-1 in the hypoxic human fetus. However, in the human fetus, zonation of IGFBP-1 and the role of hypoxia in its distribution within the fetal liver in vivo remain to be determined.
It is likely (and indeed fortunate) that the developing fetus is not exposed to the acute changes in O2 tension and profound changes in magnitude of pO2 that were used in the current experimental design for hepatocytes cultured in vitro. Of note is the lack of IGFBP-1 mRNA up-regulation with 10% O2, compared with the observed up-regulation at 2% O2. The precise mechanism underlying this observation remains unclear. We have recently found that IGFBP-1 levels in umbilical artery cord blood are inversely correlated with those of pO2, with the greatest effect seen with pO2 less than 13 mm Hg (J. Verhaege et al, unpublished data). Also, we previously reported elevated IGFBP-1 levels in human babies at term who had evidence of prolonged and profound hypoxia in utero, compared with those who had transient and reversible episodes of hypoxia (e.g. cord compression during labor and delivery) (28). Thus, duration of exposure and magnitude of the hypoxic insult are likely determinants of IGFBP-1 up-regulation in the fetal liver. In addition, the responsiveness to a hypoxic insult of isolated, cultured fetal hepatocytes in vitro may differ from that of hepatocytes in vivo.
A limited survey of the hypoxic response of human fetal tissues in explant culture indicates that fetal liver (and, to a much lower extent, fetal kidney), but not fetal lung, brain, or heart, possess the capacity to increase IGFBP-1 protein and mRNA levels (Tazuke, Lu, Giaccia, and Giudice, unpublished observations). In the current study, we found that IGFBP-3 levels increased in fetal hepatocytes under hypoxic conditions. However, in our survey of other fetal tissues, IGFBP-3 does not seem to be increased in response to hypoxia (Tazuke, Giaccia, and Giudice, unpublished). It is unclear whether the elevation of IGFBP-3 in response to hypoxia, in the current study, is physiologically relevant, because IGFBP-3 has not been shown to be elevated in fetuses with in utero hypoxia and growth restriction (11). We and others have shown that the IGFBP-3 gene has hypoxia response elements and has the capacity to respond to hypoxia, as we have previously reported in HepG2 cells (28, 39).
Elevated IGFBP-1 in fetuses with in utero hypoxia and IUGR is believed to inhibit the mitogenic and metabolic actions of IGF-I under conditions of limited substrate availability. It is noteworthy, from a teleological perspective, that the fetus may protect itself from trying to use IGF-I for growth under conditions when O2 tension is low. Furthermore, although it is tempting to suggest the potential utility of IGF-I therapy for the growth restricted fetus, data presented herein and elsewhere suggest that increasing placenta perfusion (and thus oxygenation) is likely to be critical to establishing homeostatic mechanisms in the fetus for normal growth. Whether elevation of IGFBP-1 in babies with IUGR caused by UPI and in utero hypoxia is an epiphenomenon or whether it is fundamental to the pathogenesis of the observed fetal growth restriction are important questions whose answers await further investigation.
Acknowledgments
We gratefully acknowledge Dr. Nash for providing tissue samples. We also thank Drs. J. C. Irwin and F .J. Richard for helpful discussions and help with photography.
Footnotes
1 Supported in part by Leopoldina BMBF 9701-11 (to R.M.P.), funding
from the March of Dimes Birth Defects Foundation (to L.C.G. and
A.J.G.), and Grant HD36732 (to L.C.G. and A.J.G.). 
Received February 1, 2000.
Revised October 10, 2000.
Accepted November 30, 2000.
References
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|
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