Departments of 1 Renal Pharmacology and 2 Genetic Technology, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19403
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ABSTRACT |
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To gain further insights into the molecular mechanisms involved in acute renal failure, we have isolated a new gene from rat and human, named KSP32 (kidney-specific protein with a molecular mass of 32 kDa). KSP32 encodes a novel gene that shows little homology to other mammalian proteins. It, however, shares extensive homology with several proteins found in the nematode Caenorhabditis elegans and plants. The expression of KSP32 mRNA is highly restricted to kidney. In situ hybidization analysis revealed that the expression of KSP32 mRNA was prominent in the boundary of kidney cortex and outer medulla, exhibiting a raylike formation extending from the medulla into the cortex. Finally, KSP32 mRNA was dramatically downregulated in rat following induction of acute ischemic renal failure. Rapid loss of KSP32 mRNA expression was observed beginning at ~5 h following renal injury and mRNA levels remained depressed for at least 96 h. Both KSP32 mRNA levels as well as renal function recovered 14 days after injury. Administration of an endothelin receptor antagonist (SB-209670), known to restore renal function, significantly increased KSP32 expression.
molecular cloning; medullary rays; proximal tubules; polymerase chain reaction-select
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INTRODUCTION |
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ACUTE RENAL FAILURE (ARF) is a debilitating illness for which there is limited effective therapy (37). ARF can be induced by multiple factors including renal ischemic injury, nephrotoxic drugs or by other glomerular and tubular-interstitial diseases. ARF is associated with acute cellular damage in the proximal and distal tubules followed by tubular degeneration and necrosis, leading to decreased glomerular permeability, tubular leakage and impaired renal functions (1, 2, 24, 27, 38).
Experimentally, renal artery occlusion (43) and toxic compounds such as mercuric chloride and folic acid (25) have been used in rodents to induce acute renal failure. Renal artery occlusion in rat consistently produces acute renal failure as evidenced by a rapid decrease of glomerular filtration rate (GFR) and oligouria. Although the precise mechanisms for cell damage and necrosis are not clear, ischemic shock is thought to play an important role in the initial insult to proximal tubule cells (4, 23). The site of injury is believed to be at the S3 segment of the proximal tubule and the ascending limb of the loop of Henle. Loss of epithelial cell polarity is an important early hallmark (26, 29). Necrosis and apoptosis in epithelial cells are generally recognized to play important roles in the rapid loss of glomerular filtration (22).
A number of peptide hormones have been implicated in the etiology of the onset of ARF (12, 14). In particular the role of vasoconstrictors such as endothelin (ET) has been extensively studied (5, 10). Infusion of ET-1 causes reduction of renal blood blow (RBF) and GFR, while administration of ET-1 antagonists help restore renal function in animal models of ARF (10). Other peptide hormones such as atrial natriuretic peptide (ANP) and IGF-1 have also been shown to be effective in improving renal function in animal models (15, 18, 40). In clinical trials, however, neither IGF-1 nor ANP has been shown to be beneficial (3, 14, 37). Thus far, no effective drug therapy exists for ARF.
We have undertaken a systematic effort to isolate and characterize kidney-specific genes. By using a recently available PCR-select technique (7), we have isolated a pool of genes that are expressed predominantly in renal tissue and have discovered one of these genes is highly regulated in a rat model of acute ischemic renal failure. In this paper, we describe the isolation and characterization of this gene, termed kidney-specific protein 32 (KSP32).
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MATERIALS AND METHODS |
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Materials.
A PCR-select subtraction kit was obtained from Clontech (Palo Alto,
CA). Nylon membrane (Biotran) was purchased from ICN Biotechnologies (Costa Mesa, CA). [-32P]dATP (3,000 Ci/mmol) was
purchased from NEN-DuPont (Boston, MA). Oligo(dT) agarose was obtained
from Pharmacia Biotech (Piscataway, NJ). Routine molecular cloning and
sequence analyses were performed as previously described
(35). Reagents for subcloning and sequencing were
purchased from Promega (Madison, WI). PCR reagents were obtained from
Perkin-Elmer (Norwalk, CT). Random priming labeling kits were obtained
from Promega. Mice poly(A)+ mRNA blot was purchased from
Clontech. For in vivo experiments, SB-209670 was synthesized at
SmithKline Beecham Pharmaceuticals (King of Prussia, PA) and was
prepared in 5% dextrose (vehicle). Computer programs used to analyze
the protein sequences include BLASTN, BLASTp, and Lasergene-DNA star programs.
RNA isolation.
RNA from wild-type Sprague-Dawley rat (SD) tissues (brain, heart,
liver, lung, intestine, spleen, pancreas, and kidney) was isolated as
described (35). Briefly, 1-5 g of tissue were
homogenized in 20 ml of homogenization buffer (4 M guanidium
thiocyanate, 25 mM Na citrate, 0.5% sarcosyl, and 0.1 M
beta-mercaptoethanol) by using a Polytron (Kinematica) homogenizer for
30 s at the highest speed (~10,000 rpm). Homogenized tissue
samples were then centrifuged at 5,000 g for 20 min to clear
the debris. Supernatants were then carefully added to a 9-ml Beckman
ultracentrifuge tube (no. 361623) that contained 4.5 ml of CsCl
solution (5.7 M CsCl, 25 mM Na citrate, 0.1 M EDTA). Tubes were sealed
and spun at 50,000 rpm (Ti 70.1 rotor, Beckman) for 24 h. RNAs
were pelleted at the bottom of the tube and were harvested by using
70% ethanol wash. RNAs were then dissolved in diethyl
pyrocarbonate-treated water and frozen at 80°C until use.
mRNA isolation. mRNA was isolated by using oligo(dT) agarose column as described (35). Generally, 1-2 mg of RNA from different tissues were used to isolate up to 20 µg of mRNA.
PCR-select procedure. PCR-select was performed as described in the manufacturer's instructions (Clontech). Briefly, 5 µg of mRNA were used for reverse transcription. Five micrograms of kidney mRNA were used to generate renal tester cDNA. For the rat kidney driver pool (see RESULTS), 1.25 µg of mRNA each from liver, brain, muscle, and intestine were used. For both driver and testers, a total of 5 µg mRNA were used to synthesize first and second cDNA strands. The double-stranded cDNA was subsequently digested with Dpn II restriction enzyme to produce the driver cDNA. Kidney cDNA driver and pooled four-tissue cDNA drivers were then ligated to either adaptor 1 or adaptor 2 according to the manufacturer's instruction. The pooled four-tissue cDNA driver was then used as countersubtraction to verify the subtraction efficiency. After ligation with adaptor 1 and adaptor 2, two rounds of hybridization were performed by using excess unligated driver cDNA. Two rounds of PCR (P1 and P2) were performed to amplify the subtracted and enriched cDNA species. After PCR amplification, the mixtures were analyzed on 2% agarose gels by hybridization to cDNA probes from kidney or the four-pooled tissues. If enrichment was observed, the PCR mixture was ligated to either pPCR2.1 or pPCR-TOPO (Invitrogen, Carlsbad, CA), and colonies were screened with cDNA probes.
5' and 3' RACE. Rapid amplification of cDNA ends (RACE 5' and 3') to clone the full coding sequence of KSP32 was performed by using the cDNA templates (Marathon cDNA) purchased from Clontech. Internal primers were coupled with the 5' or 3' marathon primers to PCR the KSP32 cDNA. PCR products were cloned into TA cloning vector (Invitrogen) and sequenced.
Subcloning, sequencing, and homology analyses. Cloning of PCR fragments was performed by using PCR2.1 or PCR-TOPO vectors according to the manufacturer's instructions (Invitrogen). After transformation and plating of bacteria onto IPTG-X-Gal dishes, white colonies were selected and restreaked onto two duplicated ampicillin dishes, with one dish having grided nylon membrane. After overnight growth, both dishes contained colonies, and nylon membranes were lifted to perform bacterial colony hybridization as described (35). Positive hybridizing clones were picked from the duplicate dish, and their DNA was isolated and sequenced. Homology searches were performed by using BLASTN, BLASTX, FASTN, and FASTP (NCBI BLAST server, National Institutes of Health, Bethesda, MD).
Northern analyses. Ten micrograms of RNA from various rat tissues were separated on 1% agarose formaldehyde gels and transferred to a nylon membrane. After cross-linking, the nylon membrane was hybridized with random primed 32P-labeled cDNA probes as described (35).
Animal preparation. All procedures were approved by the Institutional Animal Care and Use Committee (SmithKline Beecham Pharmaceuticals) and were in accordance with National Institute of Health Guidelines for the care and use of animals. Young (6-wk-old) male SD were obtained from Charles River Laboratories (Wilmington, MA). After quarantine, the right kidney was removed from ~50% of the rats under isoflurane anesthesia. A 2-wk recovery period was allowed for the hypertrophy of the remaining kidney. Vascular catheters were implanted into the abdominal aorta and inferior vena cava 2-4 days before the experiments. The rats were housed at all times in a light-controlled room with a 12:12-h light-dark cycle and were allowed ad libitum access to food and water.
Experimental protocol. For the occlusion of the renal artery, the rats were anesthetized with pentobarbital (40 mg/kg iv). The left renal artery was exposed through a small flank incision and occluded for 50 min with a nontraumatic clamp. Body temperature was maintained at 37°C throughout the occlusion. Three rats underwent the surgical procedure without the occlusion of the artery and were used as sham. The incision was closed after the removal of the clamp, and the rats were returned to their home cage to recover. The rats were divided into two experimental groups.
Group 1: Short-term recovery after ischemia (6 rats). The left kidney was removed under ether anesthesia at 5 or 24 h after ischemia. Then the rats were killed with an overdose of pentobarbital.
Group 2: Prolonged recovery after ischemia (34 rats).
One day after ischemia, the rats underwent 3-h iv infusion of either
vehicle, or the mixed ET-1 receptor antagonist SB-209670 (30 µg · kg1 · min
1). The
sham animals received only vehicle. The kidney was harvested and the
rats were killed in three groups, on the 2nd, 4th, or 14th day after ischemia.
Dissection of kidney and separation of glomeruli and tubular cell fractions. Glomeruli and tubular cell fractions were prepared by using a sieving technique (8, 9). Briefly, kidneys were removed from male SD (300-350 g, Charles River) and immediately placed in cold dissection buffer containing (in mM) 137 NaCl, 5 KCl, 1 CaCl2, 1.2 MgSO4, 5.5 glucose, 10 sodium acetate, 3 lactate, and 20 HEPES (pH 7.4). The kidneys were decapsulated and bisected. Cortex, pelvis, inner medulla, and outer medulla portions were excised by using razor blades. For glomeruli preparation, the cortex and outer medulla were minced into a fine paste with a razor blade and pressed through a 230-µm stainless steel sieve. The resulting material was suspended in HEPES buffer and forced through a 20-gauge needle to further break up the tissue fragments. The suspension was then passed through sieves of decreasing pore sizes of 140, 104, and 75 µm. The glomeruli, which were trapped on the 75-µm sieve, were washed from the sieve and pelleted at 1,500 g for 5 min. The flow through and materials retained at the larger sieve were also collected and RNA was prepared from them. The purity of the glomeruli was checked by light microscopy.
In situ RNA analyses.
In situ RNA hybridization analyses were performed as in Laping et al.
(21) with minor modifications: 15-µm sections of frozen kidney were mounted on RNase-free slides by using a cryostat. Sections
were fixed in 4% paraformaldehyde for 30 min. Sections were then
treated with 0.25% acetic anhydride in 0.1 M triethanolamine for 10 min, dehydrated in an ethanol series, and incubated with 0.3 ng · µl1 · kb 35S-labeled
KSP32 cRNA probe
1 in hybridization solution [50%
formamide, 4× standard sodium citrate (SSC), 5× Denhardt's, 10%
dextran sulfate, 1% SDS, and 250 µg/ml yeast transfer RNA]
overnight at 50°C under parafilm slips. Parafilm was removed
in 4× SSC and nonspecific hybridizations were removed by incubating
slides in 0.025 mg/ml RNase A (in 0.5 M NaCl and 0.05 M phosphate
buffer) at 37°C for 30 min. The wash was performed at 60°C for 30 min (50% formamide, 0.5 M NaCl, and 50 mM sodium phosphate, pH 7.4).
Dehydrated slides were dipped in nitroblue tetrazolium-2 emulsion
(Kodak) and exposed for 30 days. Developed slides were counterstained
with hematoxylin and eosin.
Antibody generation and Western analyses. A glutathione-S-transferase (GST)-KSP32 fusion protein was constructed by using PCR to insert KSP32 cDNA fragment into pGEX-2tk vector in frame. The transformed Escherichia coli (BL21) was harvested and used to generate and purify soluble fusion GST-KSP32 protein according to instructions (Pharmacia Biotech, Piscataway, NJ). Purified GST-KSP32 was injected into rabbit for polyclonal antibody. Serum bleed from the rabbits was used for the Western blot by using 1:1,000 dilutions. Enterochromaffin-like (ECL) Western blot procedure was used to visualize KSP32 protein (Amersham, Arlington Height, IL).
Total soluble kidney protein was prepared by standard procedures (35). Briefly, whole kidney was homogenized in rat intrapulmonary arteries lysis buffer containing 1% Triton-100, 1% Na deoxycholate, 0.1% SDS, 10 mM Tris · HCl, pH 8.0, 140 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, and 5 mM iodoacetamide. After centrifugation at 10,000 g at 4°C for 1 h, supernatants were collected and protein concentrations were measured by using Bio-Rad DC reagents (Bio-Rad, Hercules, CA). Fifty micrograms of total kidney proteins were separated on a SDS-NU-PAGE gel (4-12% gradient; Novex, San Diego, CA) and transferred to polyvinyl difluoride member for Western blot.Statistical analysis. Statistical analysis was performed by using the Microsoft Excel (version Excel 97) program. ANOVA (single factor) was used followed by Tukey's t-test for multiple comparisons (6).
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RESULTS |
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Molecular cloning of KSP32. A systematic search for rat kidney-specific genes was performed by using a PCR-select subtraction cloning strategy (17). Approximately 200 clones were obtained by using this procedure. One of these clones, R86, did not reveal any significant homology to other rat or human proteins in the database. Northern blot analyses indicated that the mRNA corresponding to R86 was highly kidney specific. To obtain full-length cDNA clones for R86, 5' and 3' RACE was performed to obtain a full coding sequence. This gene was subsequently named KSP32 since it is expressed almost exclusively in the kidney (see below). To obtain a human cDNA clone, a human kidney cDNA library was screened with the rat cDNA probe at lower stringency and the clones obtained were sequenced. A full-length cDNA clone for human KSP32 was verified by sequencing.
Rat and human KSP32 is a 285-amino acid protein with high homology to each other (96% similarity) (Fig. 1, A and B); however, searching the database with KSP32 revealed only four proteins with limited homology. One is an open reading frame from the Caenorhabditis elegans genome (accession no. AAB92032), identified by the genome sequencing project. Two putative proteins from Arabidopsis thaliana were also identified (accession no. CAB38955 and CAC62136). In addition, a gene isolated from Pinus radiata (Monterey pine) that was implicated in embryonic genesis of the pine (accession no. AAC05150) also showed significant similarity to KSP32 (Fig. 1C). The homology of KSP32 and these four proteins are concentrated in the amino acid range 30-280, with a 60-70% similarity to each other. The C. elegans protein has a longer NH2-terminal extension that is not found in other proteins including KSP32 (Fig. 1C). The C. elegans protein also has a much shorter COOH terminus. Blocks of highly conserved regions are clearly identifiable (regions A, B and C in Fig. 1C). All of the three regions contain aromatic amino acid residues (residues W, H, F, or Y) in the center of the region. It is currently unclear what the significance of these structure homologies are, however, the strong evolutionary conservation suggests a fundamental biological function for this protein.
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Expression of KSP32 mRNA in tissues.
Tissue distribution of KSP32 in both human and rat was examined by
Northern blot analyses. In both human and rat tissues, KSP32 expression
was found predominantly in kidney (Fig.
2A). Longer exposure of the
X-ray film revealed much lower levels (~1-5% of kidney) of
KSP32 mRNA in rat liver (Fig. 2A). The mRNA corresponding to
KSP32 is ~1.4 kb in length.
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In situ analyses of KSP32 expression.
35S-labeled anti-sense KSP32 RNA probe gave a very strong
signal on whole kidney sections while the sense strand did not give any
signals (Fig.
3B). The
expression of KSP32 mRNA was predominant at the corticomedullary
junction in both longitudinal and cross sections (Fig. 3B)
and in particular a raylike expression pattern is clearly visible in
cross section. The signal was totally absent in the inner medulla and
was sporadic in the outer cortex region. Prominent expression was
localized in the inner stripe of the outer medulla as well as the outer
stripe and medullary rays. This expression pattern agrees very well
with the Northern blot analyses performed with the fractionated kidney
(Fig. 3, B and C).
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Regulation of KSP32 expression in ischemic-induced renal failure.
Because proximal and distal tubule were severely impacted in ARF, we
investigated whether KSP32 expression was affected in a rat model of
ischemic ARF. Northern blot analyses (Fig.
4B) revealed
that KSP32 mRNA was rapidly and dramatically downregulated in
uninephrectomized rats upon ischemic renal injury. The kidney KSP32 mRNA was ~50% of normal levels 5 h following ischemic
injury and reached 5% of normal levels after 24 h. Serum
creatinine levels were increased at 5 h (from 0.5 to 1.9 mg/dl)
and at 24 h (to 4.5 mg/dl) (Fig. 4A). These results
suggest that the decline of KSP32 mRNA closely reflected the decrease
of renal function in ARF.
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In situ analyses of rat KSP32 mRNA expression after ischemic renal
injury.
Consistent with the Northern blot analyses, whole-mount exposure of
kidney sections following hybridization to 35S-labeled
KSP32 rRNA probe indicated that KSP32 expression was dramatically
reduced upon renal injury (Fig.
5A). A
slight but consistent recovery of KSP32 mRNA expression was observed in
SB-209670-, but not in vehicle-treated animals at 96 h. At 14 days, both SB-209670- and vehicle-treated rats demonstrated significant
expression of KSP32 mRNA; however, the expression of KSP32 mRNA in
SB-209670-treated rats was more intense around the medulla/cortex
boundary and its formation resembled that of normal kidney (Fig.
5A). In contrast, vehicle-treated rats had a
scattered-expression renal pattern for KSP32 mRNA (Fig. 5A).
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DISCUSSION |
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In this paper we describe the cloning and characterization of a novel renal-specific gene, termed KSP32, and its regulation in a rat model of ischemic renal failure. The KSP32 gene encodes a protein of ~32 kDa with little apparent homology to known mammalian proteins. Four proteins found in C. elegans, P. radiata, and A. thaliana, however, demonstrate a high degree of homology to KSP32. In addition, extensive homology (96% similarity) between the rat and human KSP32 suggests an important evolutionarily conserved function. Our data also indicate that the expression of KSP32 was highly restricted to the kidney. Only a limited amount (<1% of the amount found in kidney) of KSP32 was detected in the liver. The expression profile suggests a unique role for the protein in kidney function. In situ hybridization analyses revealed that the site of expression for KSP32 was primarily in the boundary of the cortex and outer medulla, especially within the medullary rays. Finally the expression of KSP32 was dramatically downregulated in a rat model of ischemic renal failure induced by renal arterial occlusion.
Gene regulation during ARF has been extensively documented (28, 31-33). Many genes have been found to be elevated, while only a limited number are suppressed (32). Two examples of genes that undergo downregulation in ARF are prepro-EGF mRNA (34) and the Tamm-Horsfall protein (THP) (33, 39). The kinetics of the reduction for EGF and THP mRNA is similar to KSP32: it begins immediately following injury and reaches a basal level at ~24 h. Several other proteins, BMP-7 (36) and IGFBP binding proteins-3 and -5 (16), have also been shown to be modestly reduced. It is unclear what role such reduction of the gene expression plays in renal failure. One hypothesis (33) is that these genes are downregulated to permit a new round of cell replication events to repair the damage. Thus the reduction of these genes is needed to "dedifferentiate" cells and allow them to reenter the cell cycle, eventually leading to repair of the tubules and restoration of renal function. Another possibility is that these genes, especially KSP32, are required for maintaining the cell polarity of the differentiated renal tubule cells in the proximal region in the outer medulla and cortex. Damage in these areas leads to cell necrosis and/or cell apoptosis. Thus the loss of KSP32 mRNA expression may be a direct reflection of proximal tubular cell damage, either by necrosis or by apoptosis in situ.
The hypothesis that KSP32 mRNA is decreased due to the loss of tubular cells is consistent with its expression pattern in kidney. In situ analyses of KSP32 mRNA distribution revealed that the KSP32 is primarily found in the outer medulla and outlining a ray-like formation into cortex. This formation bears a remarkable resemblance to the medullary rays that reside in the outer stripe of the outer medulla. This is the region that contains most of the proximal and distal tubules. These tubules are severely damaged in the early phase of ARF (4, 23). Since the expression of KSP32 in renal failure correlated very well with renal function, it is likely that the expression of KSP32 reflects the integrity of a certain subset of important renal proximal and/or distal tubular cells that are susceptible to ischemic renal injury. Inspection of the kidney sections 24 and 96 h after injury indicated severe damage to the tubules, as evidenced by the low cellularity and void spaces. Cell necrosis and apoptosis, especially tubular cells, during ischemic ARF has been well documented (13, 22, 30, 41). It is during this period that KSP32 expression was completely lost (Figs. 4 and 5). Thus it is possible that KSP32 expression reflects the integrity of the renal tubules and damage to the tubular cells caused the downregulation or loss of KSP32.
Despite an extensive search, KSP32 was not found in several commonly used renal epithelial cell lines as well as cultured primary renal proximal tubule and cortical epithelial cells. The lack of KSP32 expression in cultured kidney cells was a surprise and a hindrance to studies of the physiological role of this protein. These observations may highlight the possibility that certain in vivo factors or cell-cell interactions may be required for maintaining KSP32 expression. Preliminary results by using kidney organ cultures also confirm that KSP32 expression is rapidly (<24) lost once the kidney is dissected. These in vitro results are reminiscent of the downregulation of KSP32 observed in the in vivo rat ischemic kidney models.
In summary, we have characterized a novel renal-specific gene termed KSP32. KSP32 shares little homology with other proteins in GenBank and its localization in renal medulla suggests a role in proximal tubular function. Expression of KSP32 is highly regulated in a rat model of ischemic ARF. Dramatic downregulation is observed with 24 h of ischemic shock and the recovery of the KSP32 expression correlates with the recovery of renal functions.
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ACKNOWLEDGEMENTS |
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We wish to thank Drs. Nicholas Laping, Laura Fitzgerald, and Richard Edward of the Department of Renal Pharmacology for helpful discussions.
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FOOTNOTES |
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Address for reprint requests and other correspondence: E. Hu, Rm 25-2088, SmithKline Beecham Pharmaceutical, 709 Swedeland Road, King of Prussia, PA 19403 (E-mail: Erding_Hu-1{at}sbphrd.com).
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.
Received 24 September 1999; accepted in final form 13 April 2000.
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