Department of Pediatrics, Stanford University School of Medicine, Palo Alto, California 94305
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ABSTRACT |
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Extracellular glutathione peroxidase (EGPx) is
a glycosylated selenoprotein capable of reducing hydrogen peroxide,
organic hydroperoxides, free fatty acid hydroperoxides, and
phosphatidylcholine hydroperoxides. We found that human large
intestinal explant cultures synthesize EGPx and cellular glutathione
peroxidase (CGPx) and secrete EGPx. The level of EGPx mRNA
expression relative to -tubulin was similar throughout the mouse
gastrointestinal tract. EGPx mRNA transcripts have been localized to
mature absorptive epithelial cells in human and mouse large intestine.
Western blot analysis of mouse intestinal protein has demonstrated the
presence of EGPx protein in the small intestine, cecum, and large
intestine, with the highest protein levels found in the cecum.
Immunohistochemistry studies of human large intestine and mouse small
and large intestine sections demonstrated the presence of EGPx protein
within mature absorptive epithelial cells. In human large intestine and
mouse small intestine, EGPx protein is also present in the
extracellular milieu. These results suggest a role for EGPx in
protection of the intestinal tract from peroxidative damage
and/or in intercellular metabolism of peroxides.
antioxidant; epithelial cells; oxidative damage; peroxides; selenium
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INTRODUCTION |
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SELENIUM-DEPENDENT glutathione peroxidase (GPx) is an antioxidant enzyme that detoxifies hydrogen peroxide and lipid hydroperoxides using reduced glutathione and thus protects biomembranes and essential cellular components against oxidative damage (16). Extracellular GPx (EGPx) is genetically distinct from the classical cellular GPx (CGPx), membranous phospholipid hydroperoxide GPx (28), and an intestinal form of GPx (GPx-GI) (11). EGPx is capable of reducing hydrogen peroxide, organic hydroperoxides, free fatty acid hydroperoxides, and phosphatidylcholine hydroperoxides (14, 32-22). EGPx has been found in plasma, milk, amniotic fluid, exocoelomic fluid, and fluid from lavaged lung (3, 6, 7, 19). In adults, most of the blood plasma EGPx activity is derived from the kidney with additional sites of synthesis of EGPx mRNA in the lung, heart, and intestine (2, 5, 19). Elucidating the sites of synthesis and secretion of EGPx is necessary to gain better understanding of the function of this unique selenoglycoprotein. The human colon adenocarcinoma cell line Caco-2 is one of several cell lines that synthesizes and secretes EGPx (4). In addition, mouse intestine is also a site of synthesis of EGPx mRNA (19, 24). This study describes further investigation of the synthesis of EGPx in the intestine.
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EXPERIMENTAL PROCEDURES |
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Animals. Adult BALB/c mice, at ~60 g, were the source of all experiments involving mouse intestine.
Human tissue. All described experiments involving human intestine were performed using nontumorous adult human intestinal tissues derived from material that was removed as a part of resection surgery.
Culturing of human intestinal explants and exposure to metabolic
labeling with [35S]methionine.
Human intestinal explants were cultured by modification of the method
described by Autrup et al. (1). The specimens were cleansed of
mesentery and cut into 5 × 5 mm pieces. Explants were placed in
24-well plastic tissue culture dishes coated with rat tail tendon
collagen, with the epithelium facing the gas-medium interface and the
serosa of the intestine resting on the collagen-coated dish. Explants
were stabilized for 4 h in medium that contained DMEM (Mediatech,
Herndon, VA) with 20% fetal bovine serum (FBS) (HyClone Laboratories,
Logan, UT) supplemented with 1 × 107 M sodium selenite.
Explants were placed in stabilizing medium within 30 min after surgical
removal. The tissue was cultured at 37°C in a humidified incubator
(Forma Scientific, Marietta, OH) with an environment of 7.5%
CO2-92.5% air.
Immunoprecipitation of metabolically labeled selenoproteins from
intestinal explants in culture and culture medium.
Immunoprecipitation and analysis of immunoprecipitates were performed
by a modification of the method of Avissar et al. (2). Metabolically
labeled intestinal tissues were homogenized using a Tissue Tearor
(Biospecs Products, Racine, WI) in 0.2% SDS, 2 mM
N-ethylmaleimide (NEM), 2 mM
iodoacetic acid (IAA), 4 mM phenylmethylsulfonyl fluoride (PMSF), and 4 mM EDTA in 0.1 M Tris buffered to pH 8.3. The cell
extracts were centrifuged for 15 min at 2,500 rpm, and the supernatants
were saved. An equal volume of a solution containing 1% Nonidet P-40,
1% deoxycholate, and 0.1 M Tris · HCl (pH 8.3) was
used to wash the homogenizer and then added to the saved supernatants to bring the constituents to a final concentration of 0.1 M Tris, 0.1%
SDS, 1 mM NEM, 1 mM IAA, 2 mM PMSF, 2 mM EDTA, 0.5% Nonidet P-40, and
0.5% deoxycholate. The culture medium was centrifuged, and the
cell-free supernatant was supplemented to give a final composition of 1 mM IAA, 1 mM PMSF, 0.1% SDS, and 1 mM NEM. All samples were saved at
70°C until further processing.
Isolation and hybridization of total RNA.
Alternating 2-cm intestinal sections from mice were dissected from the
proximal duodenum to the distal colon. Sections were rinsed with PBS
for mRNA isolation. Total RNA was isolated using a combination of the
Ultraspec RNA isolation system (Biotecx, Houston, TX) and a modified
Chomczynski and Sacchi method (27). We electrophoresed 10 µg of total
RNA in 1.5% agarose-formaldehyde gels. The resolved RNA was
transferred to positively charged Nylon membranes (Nytran Plus,
Schleicher & Schuell, Keene, NH) with 20× SSC (1× SSC is
0.15 M NaCl and 0.015 M sodium citrate, pH 7.0) and ultraviolet
cross-linked (Stratalinker, Stratagene, La Jolla, CA). Random-primed
(RadPrime labeling kit, GIBCO, Gaithersburg, MD) murine EGPx c-DNA
(clone 47-A1, a kind gift of R. Maser, University of Kansas) was
labeled with
[-32P]dCTP (sp act
3,000 Ci/mmol, DuPont NEN) to specific activities >1 × 108 cpm/µg. The blots were
prehybridized and hybridized in 5× Denhardt's solution, 50%
deionized formamide, 10% dextran sulfate, 250 µg/ml salmon sperm,
1% SDS, and 2× SSC at 42°C overnight. The blots were then
washed at 55°C with the most stringent washing in 0.1× SSC
and 1% SDS before exposure to a phosphor-imaging screen. The screens
were scanned and quantified using a Bio-Rad GS-505 Molecular Imager
(Hercules, CA). The blots were stripped in boiling 0.1% SDS for 1 min
and checked for background before the next probe was used. The blots
were also probed with CGPx (a gift from Dr. N. Imura, Kitasato
University, Japan) and
-tubulin using probes prepared in identical fashion.
Preparation of RNA from villus-enriched mouse small intestine.
To provide additional evidence for the localization of mRNA, we excised
~2 cm of proximal ileum from the mouse. Using the polished wide-open
end of a glass pipette, we lightly scraped away the villus epithelium
from the muscularis. RNA from the villus epithelium and muscularis
samples were prepared and probed as in the above-described procedure,
except glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was utilized in
lieu of -tubulin.
In situ hybridization. Normal human and mouse intestinal tissue were obtained and cut into pieces of ~27 mm3, fixed in 4% paraformaldehyde overnight, embedded in paraffin, and sectioned (5 µm) onto gelatin-subbed glass slides. After deparaffinization with xylene and rehydration through graded ethanol washes (100, 95, 70, 50, and 30%), sections were treated sequentially with 1 µg/ml proteinase K for 5 min, washed, and dipped in 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0) for 5 min. Sense and antisense cRNA probes were made from linearized plasmids containing EGPx and CGPx cDNA inserts by in vitro transcription in the presence of biotinylated nucleotides (NEN). Before hybridization, limited alkaline hydrolysis of RNA probes was performed to reduce transcript length to 0.1-0.3 kb (44 mM NaHCO3 and 66 mM Na2CO3, pH 10.2, for 25 min at 60°C). Sections were hybridized overnight at 50°C in a solution containing 50% formamide, 0.6 M NaCl 10 mM Tris · HCl (pH 7.5), 1 mM EDTA, 1× Denhardt's reagent, 10% PEG 8000, 50 µg/ml heparin, 0.5 mg/ml yeast tRNA, and 2 ng/µl RNA probe. Probed sections were then covered with silanized glass coverslips. After hybridization, coverslips were removed in warmed 2× SSC. The sections were washed in 2× SSC for 30 min at 50°C and treated with RNase A 20 µg/ml) in 2× SSC at 37°C for 30 min. The slides were then washed for 30 min at 50°C in 50% formamide in 2× SSC followed by 0.07% sodium pyrophosphate in 1× SSC. Slides were developed using a horseradish peroxidase-tyramide signal amplification kit (NEN), visualized with nickel-enhanced 3,3'-diaminobenzidine (DAB), and stained with hematoxylin (Biomeda, Foster City, CA).
Western blot analysis. For analysis of expression of EGPx protein, 100 mg of mouse small intestine (proximal ileum), cecum, and large intestine (proximal colon) were collected and homogenized in 10 mM Tris, 150 mM NaCl, 0.5% Triton X-100, 1% Nonidet P-40, 10 mM NaN3, 1 µg/ml leupeptin, 1 mM para-aminobenzamide, and 0.5 mM PMSF, pH 7.5. Fifty micrograms of intestinal protein were analyzed by 12.5% SDS-PAGE under reducing conditions (100 mM DTT) and electroblotted onto a membrane (Hybond polyvinylidene difluoride membrane, Amersham Life Science). The membranes were blocked with PBS containing 6% (wt/vol) nonfat dry milk at room temperature for 1 h and then incubated for 1 h with either rabbit anti-mouse EGPx antibody or rabbit anti-mouse CGPx IgG at a 1:20,000 dilution. After the membranes were washed in PBS containing 0.05% polyethylene-sorbitan monolaurate, the immunoblots were incubated with the secondary antibody goat anti-rabbit IgG-horseradish peroxidase at a 1:10,000 dilution (Pierce, Rockford, IL) for 1 h at room temperature and developed with Super Signal Western blotting substrate (Pierce). Blots were exposed to X-ray film and quantified by densitometry (PDI). Mouse plasma and kidney were used as controls.
Immunohistochemistry. Human and mouse intestinal tissue were dissected into 27-mm3 sections and placed into 4% paraformaldehyde fixative. Sections were embedded in paraffin, and 5-µm thick sections were prepared on gelatin-coated glass slides. After microwave oven heating of slides for 10 min in 0.01 M citrate buffer, pH 6.0, to abolish endogenous alkaline phosphatase activity, sections were incubated with either rabbit anti-human or -mouse EGPx antibody and rabbit anti-human or -mouse CGPx antibody at 10 µg/ml for 1 h at room temperature. After several washes with PBS, sections were sequentially incubated with biotin-sp-conjugated affinity-purified donkey anti-rabbit IgG at a 1:2,500 dilution (Accurate Chemical and Scientific, Westbury, NY) and streptavidin-alkaline phosphatase conjugate (Calbiochem) at a 1:2,500 dilution for 1 h each at room temperature. Sections were visualized with fast red (Biomeda) and counterstained with hematoxylin. Some sections were visualized with streptavidin-horseradish peroxidase at a 1:100 dilution plus nickel-enhanced DAB. When using horseradish peroxidase, the sections were first treated with 0.3% H2O2 in PBS for 30 min. Rabbit IgG was used as a control (Sigma Chemical, St. Louis, MO).
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RESULTS |
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Human large intestinal explants synthesize CGPx and EGPx and secrete EGPx. To determine whether the large intestine synthesizes and secretes EGPx, we metabolically labeled normal human large intestinal explants with [35S]methionine for 24 h. Labeled proteins were exhaustively immunoprecipitated from the explants and the medium using our previously described antibodies and analyzed by SDS-PAGE under reducing (100 mM DTT) conditions. These rabbit polyclonal antibodies were raised against purified CGPx and EGPx and are specific and mutually non-cross-reacting (28). Under reducing conditions, immunoprecipitated EGPx and CGPx have apparent molecular masses of 24 and 23 kDa, respectively. As can be seen in Fig. 1A, EGPx and CGPx are present in the explants and EGPx only is present in the medium. The presence of radioactive bands at these specific locations demonstrates that the explants synthesized both EGPx and CGPx, but only EGPx was secreted. Trace amounts of a band immunoprecipitated by anti-CGPx IgG from the medium of the explants are probably due to some cell lysis during culture of these explants (Fig. 1). The ratio of medium to explant for EGPx is much greater than the ratio of medium to explant for CGPx (Fig. 1B). This difference indicates that the amount of EGPx in the medium is due to secretion and not to cell lysis or leakage.
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EGPx and CGPx mRNA is expressed throughout mouse gastrointestinal
tract.
To determine the expression pattern of EGPx and CGPx mRNA along the
gastrointestinal tract, we isolated total RNA from various mouse
intestinal sections and analyzed it using Northern blot analysis. To
ensure that equal amounts of mRNA were being analyzed, the same blots
were probed with -tubulin and the levels of both EGPx and CGPx were
normalized to the level of
-tubulin. As can be seen in Fig.
2, the level of EGPx mRNA expression was
similar throughout the mouse gastrointestinal tract as measured by
Northern blot analysis when normalized to
-tubulin. CGPx mRNA
expression, when normalized to
-tubulin (Fig. 2), appears to
increase from the proximal to the distal end of the gastrointestinal
tract. Thus EGPx mRNA expression relative to CGPx mRNA expression is higher in the proximal end of the gastrointestinal tract.
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Localization of EGPx and CGPx mRNA in human and mouse large intestine. Using biotin-labeled anti-sense EGPx and CGPx cRNA probes, we found that only a subset of cells in the large intestine contained EGPx and CGPx transcripts. In human and mouse large intestine, EGPx and CGPx transcripts were localized predominantly to the mature absorptive epithelial cells (Fig. 3). In addition, in mouse small and large intestine, CGPx transcripts were localized to the muscularis (data not shown). Using these conditions and probes, we were unable to demonstrate the presence of either EGPx or CGPx mRNA in human or mouse small intestine (data not shown). To provide additional evidence for the localization of EGPx mRNA, the villus epithelium was gently scraped away from the muscularis in mouse small intestine. EGPx and CGPx mRNA were measured by Northern blot analysis. To ensure that equal amounts of mRNA were being analyzed, the same blot was probed with GAPDH and the levels of both EGPx and CGPx were normalized to the level of GAPDH (Fig. 4). EGPx mRNA was four to five times greater in the samples enriched for the villus epithelium compared with samples enriched for the muscularis (Fig. 4). Using the same blot and procedure, we detected CGPx mRNA to a similar degree in both the villus epithelium and muscularis (Fig. 4). These data support the localization of EGPx mRNA predominantly to the villus epithelium.
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Presence of EGPx and CGPx protein in mouse intestine by Western blot. EGPx and CGPx protein levels in the mouse gastrointestinal tract (proximal ileum segment of small intestine, cecum, and proximal colon segment of large intestine) were determined by Western blot using polyclonal antibodies raised against peptides specific for EGPx or CGPx. As shown in Fig. 5A, EGPx protein was present in all three sampled intestinal sections. Within the gastrointestinal tract, the highest amount of EGPx protein was present in the cecum (Fig. 5B). In comparison, CGPx protein was also present in the three sampled intestinal sections (Fig. 5A).
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Localization of EGPx and CGPx protein in human and mouse intestine. EGPx and CGPx protein localization in the mouse and human gastrointestinal tract was determined by immunohistochemistry using affinity-purified antibodies directed against peptides specific for EGPx or CGPx. In human large intestine, EGPx protein is concentrated in mature absorptive epithelial cells and beneath the basolateral membrane of the epithelial layer, consistent with its presence in the extracellular milieu. In contrast, CGPx protein is found only in the mature absorptive epithelial cells and throughout the lamina propria (Fig. 6, A and B). In the mouse large intestine, EGPx and CGPx proteins are predominantly located in the mature absorptive epithelial cells (Fig. 6, C and D). In the mouse large intestine samples, it could not be determined if EGPx protein was present in the space consistent with the extracellular milieu (Fig. 6C). In additional experiments, we were able to demonstrate in mouse small intestine that EGPx protein was present in extracellular compartments, whereas CGPx protein was present only in cellular compartments (Fig. 7, arrows). In addition, CGPx protein was also found in the muscularis of mouse small and large intestines (data not shown).
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DISCUSSION |
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EGPx has been shown to be synthesized and secreted by the human colon adenocarcinoma cell line Caco-2, in addition to kidney and liver cell lines (4). In this study, we have demonstrated that explants of resected normal human large intestine synthesize EGPx and CGPx and secrete EGPx. Northern blots of mouse intestinal mRNA have demonstrated that EGPx transcripts are found throughout the gastrointestinal tract. In situ hybridization studies have shown that EGPx mRNA transcripts are localized to mature absorptive epithelia of human and mouse large intestine. Additional verification for the localization of EGPx mRNA in the villus epithelium in mice was obtained by Northern analysis of samples enriched for villus epithelium. The scraping procedure previously described generally removes the distal villus epithelium, leaving behind the crypts with the muscularis (30). The four- to fivefold greater amount of EGPx mRNA found in the villus epithelium compared with the muscularis may underestimate the relative contribution since the technique may not have completely removed all of the villus epithelium from the muscularis. Western blot analysis of mouse intestinal protein has demonstrated the presence of EGPx protein in small intestine, cecum, and large intestine, with the highest protein levels found in the cecum. With the use of immunohistochemistry, EGPx protein has been found within mature absorptive epithelia in human large intestine and mouse small and large intestine and within the extracellular milieu in human large intestine and mouse small intestine.
Northern analysis of mouse intestinal tissue revealed similar levels of EGPx mRNA expression throughout the entire mouse gastrointestinal tract. Surprisingly, the amount of EGPx protein detected by Western analysis was not similar for the three sections of mouse intestine that were sampled. It is unclear at the present time as to whether this difference is due to changes in rates of synthesis, secretion, or degradation. We were also unable to detect any evidence of EGPx and CGPx mRNA in human and mouse small intestine during in situ hybridization. However, using the same tissue samples, we were able to detect protein by the method of immunohistochemistry. Perhaps the mRNA in these particular samples were unstable under the conditions utilized during in situ hybridization, which prohibited us from detecting the transcripts of both EGPx and CGPx.
Another member of the selenium-dependent GPx family, GPx-GI, has been identified by Chu et al. (11). This cellular selenium-dependent GPx is readily detectable in the rat gastrointestinal tract. Esworthy et al. (15) have reported that GPx-GI and CGPx activities were uniformly distributed in the rat middle and lower gastrointestinal tract and with respect to the villus-to-crypt axis. In addition, GPx-GI activity levels were found to be nearly equal to that of CGPx throughout the rat small intestine and colorectal segments. We were able to detect CGPx mRNA and protein in human and mouse intestinal epithelial cells, in agreement with recent work in rats and mice by Esworthy et al. (15).
The intestinal epithelial cells are composed of rapidly dividing stem cells, which are located in crypts, and differentiated short-lived cells, which are located in villi. It has been demonstrated that the activities of several antioxidant enzymes, including GPx, catalase, superoxide dismutase, and glutathione S-transferase, are present in the intestinal epithelium. However, the exact location of GPx enzyme activity within the mucosal epithelium has variously been reported to range from its predominance in either the distal villus (23), the crypt region (12), or both (15). The antioxidants present in the intestinal epithelium represent the first line of defense against the ingested oxidative toxins and xenobiotics. The villus epithelial cells of the intestine are directly exposed to dietary materials and may therefore be especially susceptible to oxidative damage. Dietary peroxides can be metabolically reduced to hydroxy lipids in the gastrointestinal tract (20). Our data from humans and mice have demonstrated that EGPx and CGPx mRNA are predominantly found in the villus. Their presence in the villus suggests a protective role in this region. CGPx, as an intracellular GPx, would protect the villus epithelial cells, while EGPx would provide extracellular antioxidant protection for the villus. Exogenous glutathione has been shown to protect the intestinal villus from oxidative injury (8, 9, 20, 22). Exogenous glutathione may therefore enhance the effectiveness of these different components of the GPx system (EGPx, CGPx, and GPx-GI) of the gastrointestinal tract.
Inflammatory bowel disease (IBD), such as Crohn's disease and ulcerative colitis, causes chronic injury to the gastrointestinal tract, and studies have shown that antioxidant defenses are altered in these diseases. Studies have demonstrated that the colons of IBD patients produce more oxygen free radicals compared with those of control subjects (17). Free radicals have been implicated in colonic disease, such as ulcerative colitis (25-26), and in vitro studies using enterocytes have shown the damaging effect of exposure to oxidants (10). Considerable debate still exists as to whether these free radicals are involved in the pathogenesis of tissue injury or are generated as a consequence of damaged tissue. The increase in mucosal permeability that results from injury to the epithelium may facilitate the entry of luminal components such as bacteria and bacterial products into the mucosal interstitium, which may ultimately reach the systemic circulation. The clear presence of EGPx in the colon suggests a protective mechanism for reactive oxygen species that have found their way into the extracellular milieu. It has been reported that patients with IBD have an increase in GPx activity in their plasma (17, 29). However, a direct relationship between IBD and EGPx expression in the gastrointestinal tract remains to be determined. Under ordinary circumstances, plasma GPx activity is mainly derived from the kidneys (5). However, ~5-25% of the normal plasma GPx activity appears to be due to nonrenal sources, as seen in the residual activity in the plasma of anephric patients (5, 31). We have presented data that demonstrate secretion of extracellular GPx by intestinal explants. The immunohistochemistry results have demonstrated EGPx protein located outside the epithelial cells, beneath the absorptive epithelial cells in the human large intestine and mouse small intestine, which may suggest a basolateral secretion. The secretion of EGPx by intestinal epithelia may provide extracellular protection within the intestine, in the interstitial space. Intestinal expression of EGPx might be needed as a protective mechanism against lipid peroxides found in this specific area.
This present study has demonstrated the presence of the defense enzyme EGPx in the gastrointestinal tract, and this enzyme along with others may provide protection against oxidant injury. We have demonstrated that selenium-dependent EGPx is synthesized and secreted by human and mouse intestinal epithelia. Its presence in the extracellular milieu of the intestine suggests a localized role in antioxidant defense and/or metabolism of peroxides.
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ACKNOWLEDGEMENTS |
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We thank Dr. Rodney U. Anderson for providing tissue specimens.
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FOOTNOTES |
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This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant 2R01-DK-33231.
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: D. M. Tham, Dept. of Pediatrics, Stanford Univ. School of Medicine, 300 Pasteur Drive, Rm. S308, Stanford, CA 94305-5208.
Received 16 June 1998; accepted in final form 31 August 1998.
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