1 Orthopaedic Microsurgery Research Laboratory, Department of Surgery, 2 Orthopaedic Cell Biology Laboratory, and 3 Howard Hughes Medical Institute, Pulmonary and Cardiovascular Divisions, Department of Medicine, and Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710
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ABSTRACT |
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This study examined mRNA and protein expressions of neuronal (nNOS), inducible (iNOS), and endothelial nitric oxide synthases (eNOS) in peripheral nerve after ischemia-reperfusion (I/R). Sixty-six rats were divided into the ischemia only and I/R groups. One sciatic nerve of each animal was used as the experimental side and the opposite untreated nerve as the control. mRNA levels in the nerve were quantitatively measured by competitive PCR, and protein was determined by Western blotting and immunohistochemical staining. The results showed that, after ischemia (2 h), both nNOS and eNOS protein expressions decreased. After I/R (2 h of ischemia followed by 3 h of reperfusion), expression of both nNOS and eNOS mRNA and protein decreased further. In contrast, iNOS mRNA significantly increased after ischemia and was further upregulated (14-fold) after I/R, while iNOS protein was not detected. The results reveal the dynamic expression of individual NOS isoforms during the course of I/R injury. An understanding of this modulation on a cellular and molecular level may lead to understanding the mechanisms of I/R injury and to methods of ameliorating peripheral nerve injury.
nitric oxide synthase transcription; nitric oxide synthase translation; reperfusion; sciatic nerve
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INTRODUCTION |
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NITRIC OXIDE (NO) acts as a neurotransmitter (22), and NO synthase (NOS) isozymes have been shown in the central nervous system (30), dorsal root ganglia (DRG) (7, 37), autonomic nerve fibers (22), and neuromuscular end plates (3, 21). Evidence indicates that NO is also a mediator of nerve injury (12, 41) or the physiological response to injury and may be responsible for abnormal neural activity and pain behavior in animals (39). In the peripheral nervous system, studies have focused on the effects of axonal injury, especially on neurons in the DRG and spinal cord. After ligation of nerve roots, NOS activity was bilaterally decreased in spinal cord and ipsilaterally increased in DRG (7). Peripheral nerve sectioning, however, resulted in an increase in NOS messenger ribonucleic acid (mRNA) in the corresponding DRG (37, 39), which appeared within 2 days and persisted for >2 mo after operation (37).
Alternatively, NO may be involved in regeneration of injured peripheral nerve (44). Data have shown that the three forms of NOS are overexpressed at day 2 after rat sciatic nerve ligature, reaching their greatest expressions during the 2nd wk (14). In particular, long-lasting (4 wk) NOS activity in growth cones and the coexpression of NOS and growth-associated protein (GAP-43) suggest that NO contributes to regeneration (15). However, conflicting results have also been reported. In a mouse study, an NOS inhibitor enhanced regeneration of the proximal stump of transected sciatic nerve, suggesting that NO may have a negative impact on the regeneration (43). NOS mRNA was found in motoneurons of the avulsed root segment where most motoneurons had died. However, this expression was completely inhibited in all motoneurons that regenerated into the peripheral nerve graft (40).
Numerous studies have attempted to characterize the function of NO in ischemia-reperfusion (I/R) injury of the central and peripheral nervous systems; however, no consensus has been reached (17, 30). Hypoxia induced a 10-fold increase of nNOS mRNA expression in nodose ganglia and a 2-fold increase in the cerebellum (32). However, there are no relevant studies specifically addressing the significance of NO in I/R injury of peripheral somatic nerves. We therefore undertook this study to identify the changes in mRNA and protein expressions of constitutive neuronal (nNOS), endothelial (eNOS), and inducible NOS (iNOS) in rat sciatic nerve during ischemia and during early reperfusion.
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MATERIALS AND METHODS |
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Both sciatic nerves from 66 male Sprague-Dawley rats weighing 250-300 g were isolated and divided into two groups. In group 1 (ischemia, n = 26), a 1.5-cm segment of one sciatic nerve underwent 2 h of in situ warm ischemia to match the ischemic duration of the I/R group. Complete ischemia was achieved using two 6-0 nylon sutures to ligate the nerve at the proximal and distal ends of the segment. In group 2 (I/R, n = 40), a specially designed device was applied to produce a low-load crush injury, which mimicked an I/R injury (4, 5). In this group, a 5-mm segment of one sciatic nerve in each animal was subjected to a 100-g crush load for 2 h to simulate ischemia, followed by 3 h of reperfusion, after which the nerves were harvested. In each group, the opposite sciatic nerve of each rat was used as a normal control. The surgical procedures were based on those of Chen et al. (4).
mRNA Isolation and cDNA Synthesis
Procedures were based on our earlier studies (34). Poly(A)+ RNA was extracted from the harvested nerves by using Dynabeads oligo(dT)25 (Dynal, Lake Success, NY) according to the manufacturer's instructions. Briefly, the nerve was lysed in lysis/binding buffer that contained 100 mM Tris · HCl, pH 8.0, 500 mM LiCl, 1% lithium dodecyl sulfate (LiDS), 5 mM dithiothreitol, and 10 mM EDTA (Sigma, St. Louis, MO) and sonicated by an ultrasonic processor (Heat Systems, Farmingdale, NY). The lysate was centrifuged to remove debris and was then combined with the Dynabeads oligo(dT)25. Dynabeads were washed twice in a washing buffer containing 10 mM Tris · HCl, pH 8.0, 150 mM LiCl, 0.1% LiDS, and 1 mM EDTA and once with washing buffer without LiDS. Poly(A)+ RNA was eluted from beads in diethylcarbonate-treated water at 67°C, and RNA concentration was measured by using DNA Dip Stik (Invitrogen, San Diego, CA) immediately according to the manufacturer's instructions. Poly(A)+ RNA was reverse transcribed in a PCR machine (model 2400; Perkin-Elmer, Norwalk, CT). A 20-µl reaction mixture contained 2 ng of mRNA, 2.5 µM oligo(dT)16 as a primer, 5 mM MgCl2, 20 units of RNase inhibitor, 1 mM dNTPs, and 50 units of Moloney murine leukemia virus reverse transcriptase (Perkin-Elmer, Branchburg, NJ). First-strand cDNA was synthesized at 25°C for 10 min, 42°C for 15 min, and 99°C for 5 min. In a selected tube, the reverse transcriptase was omitted to control for amplification from contaminating cDNA or genomic DNA.Quantitative PCR
Quantitation of NOS mRNA levels was performed by a method that involves simultaneous coamplification of both the target cDNA and a reference template (MIMIC) with a single set of primers (35). MIMIC for nNOS, eNOS, iNOS, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were constructed using a PCR MIMIC construction kit (Clontech Laboratories, Palo Alto, CA). Each PCR MIMIC consists of a heterologous DNA fragment with 5'- and 3'-end sequences that was recognized by a pair of gene-specific primers. The sizes of PCR MIMICs were distinct from those of the native targets. The sequence of oligonucleotide primer pairs used for construction of MIMIC and amplification of NOS isoforms and GAPDH mRNA is listed in Table 1. Aliquots were taken from pooled first-strand cDNA from the same group and constituted one sample for PCR. A series of 10-fold dilutions of known concentrations of the MIMIC were added to PCR amplification reactions containing the first-strand cDNA. PCR MIMIC amplification was performed in 100 µl of a solution containing 1.5 mM MgCl2, 0.4 µM primer, PCR DIG labeling mix (200 µM dNTP), 0.002 µM Taq DNA polymerase, and 0.056 µM TaqStart antibody (Clontech Laboratories). After an initial denaturation at 95°C for 65 s, the cycle condition was 15 s at 95°C and 30 s at 60°C for 45 cycles (NOS) and for 35 cycles (GAPDH). Samples were incubated for an additional 8 min at 72°C before completion. Digoxigenin-labeled PCR products were subjected to electrophoresis on agarose gels and transferred to nylon membranes in 10× saline-sodium citrate. Membranes were air-dried, and chemiluminescent detection was carried out using a Wash and Block Buffer Kit and disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13,7]decan}-4-yl) phenyl phosphate (Boehringer-Mannheim, Indianapolis, IN). Kodak Biomax MR film was exposed to detect chemiluminescent signals. Autoradiogram band intensity was quantitated by densitometry using NIH Image software. Levels of mRNA were calculated from the point of equal density of the sample and MIMIC PCR products (Fig. 1). NOS mRNA levels were normalized with the levels of GAPDH mRNA present in each sample, which served to control for variations in RNA purification and cDNA synthesis. The relative mRNA expression of NOS in each group was compared with those from the respective normal group and reported as a percentage of normal.
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Western Blot
Sciatic nerve was homogenized in lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 0.1% SDS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.02% sodium azide, 1 mM phenylmethylsulfonyl fluoride, 10 µM leupeptin) and centrifuged at 100,000 g at 4°C for 1 h. Protein concentration in the supernatant was measured with a bicinchoninic acid kit (Pierce, Rockford, IL), and 50 µg of total protein were loaded onto an 8% SDS-polyacrylamide gel and then transferred onto nitrocellulose membrane (Micron Separations, Westborough, MA). The membrane was blocked with 5% nonfat dry milk in TBS-T (pH 7.6, 20 mM Tris base, 137 mM NaCl, and 0.1% Tween 20) at room temperature for 1 h and then incubated with monoclonal primary antibody for nNOS (1:2,000), eNOS (1:1,000), or iNOS [1:2,500; Transduction Laboratories, Lexington, KY; iNOS monoclonal antibody (MAb) was also obtained from Santa Cruz Biotechnology, Santa Cruz, CA] at 4°C overnight. After three washes in TBS-T buffer, the blot was incubated with 1:4,000 horseradish peroxidase-labeled goat anti-mouse IgG (Calbiochem, La Jolla, CA) for 1 h at room temperature. After washes in TBS-T buffer, the blot was detected with an enhanced chemiluminescence detection kit (Amersham). Kodak film was used to detect chemiluminescent signals.Immunohistochemistry
Five-micrometer longitudinal and cross sections of the nerve were air-dried at room temperature for 30 min, fixed in 4% paraformaldehyde for 10 min, and then blocked with 0.3% H2O2 in methanol at room temperature for 30 min to quench the endogenous peroxidase activity. The sections were washed for 2 min three times in PBS, incubated with 5% horse serum for 20 min to block nonspecific binding, and then incubated with mouse anti-nNOS (1:800), anti-eNOS (1:800), and anti-iNOS (1:50) monoclonal primary antibody at 4°C overnight. After they were thoroughly washed in PBS, the sections were incubated with biotinylated horse anti-mouse rat absorbed secondary antibody (1:200; Vector Laboratories, Burlingame, CA) at room temperature for 30 min and then detected with Vectastain ABC kit (Vector Laboratories). The sections were then developed with diaminobenzidene (Calbiochem) and counterstained with hematoxylin (Sigma). Mouse isotope-matched IgG was used to replace the primary antibody as a negative control.Schwann cells were identified with a rabbit anti-S-100 polyclonal antibody (1:1,200; Dako). The immunostaining procedure was the same as that described above, except the blocking buffer was 5% goat serum and the secondary antibody (1:200; Dako) was biotinylated. Rabbit isotope-matched IgG was used as the negative control.
Statistical Comparison
Values are means ± SD of three experiments. Statistical comparisons utilized one-way analysis of variance, with P < 0.05 indicating significance. ![]() |
RESULTS |
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Expressions of mRNA and Protein of Three NOS Isoforms in Normal Nerve
mRNA expression.
With the use of specific oligonucleotide primers for nNOS, iNOS, and
eNOS, the RT-PCR amplified products showed single clear bands of the
predicted sizes (Fig. 2). With the use of
restriction enzyme digestion, each of the primers gave fragments of
expected sizes based on the reported sequences. iNOS mRNA was expressed at a very low level.
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Protein expression.
In the nerve homogenate, a 155-kDa protein band identified by the nNOS
MAb comigrated with the nNOS positive control from rat brain, and a
140-kDa protein band identified by an eNOS MAb comigrated with the eNOS
positive control from human endothelial cells (Fig.
3). An iNOS MAb purchased from
Transduction Laboratories detected a protein band with an approximate
size of 160 kDa (Fig. 3); however, this was larger than that of the
iNOS positive control from macrophage (130 kDa) and could not be
confirmed with an iNOS MAb from Santa Cruz Biotechnology. The absence
of a cross-reaction between nNOS MAb and eNOS positive control, between
eNOS MAb and nNOS positive control, and between iNOS MAb and eNOS or
nNOS positive control suggested that each NOS MAb was isoform specific.
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Immunohistochemistry.
Reaction of nerve sections with nNOS MAb revealed a robust brown
staining of Schwann cells (myelinated and unmyelinated) that appeared
to be cytoplasmic. The identity of Schwann cells was verified by
anti-S-100 antibody staining. Most axons also stained, although the
staining was less intense than that observed in Schwann cells. The
myelin sheath, extracellular matrix, and blood vessels did not appear
to stain with nNOS MAb (Fig.
4A). Only the endothelial cells of the microvessels were markedly stained by the eNOS MAb (Fig.
4B), and there was no staining of the axons or Schwann
cells. Although iNOS MAb obtained from Transduction Laboratories
stained the endothelial cells and the periphery of the Schwann cells
(Fig. 4C), this positive staining was not confirmed by the
iNOS MAb from Santa Cruz Biotechnology (Fig. 4D). Negative
controls using mouse IgG or rabbit IgG, instead of primary antibody,
revealed no nonspecific staining.
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mRNA Expression of Three NOS Isoforms in Ischemic and Reperfused Nerve
At the end of 2 h of ischemia, nNOS and eNOS mRNA expressions remained at normal levels, but iNOS showed a sixfold (604 ± 150%) increase from normal sciatic nerve of the opposite limb (P < 0.001). After 2 h of ischemia and 3 h of reperfusion, nNOS and eNOS mRNA expressions diminished to 91 ± 30 and 62 ± 25% of normal, respectively. In contrast, iNOS mRNA further increased to 1,382 ± 204% of normal, a 14-fold upregulation. Compared with normal and ischemia-only groups, downregulation of eNOS mRNA expression and upregulation of iNOS mRNA expression were significant (Fig. 5).
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Protein Expression of Three NOS Isoforms in Ischemic and Reperfused Nerve
After 2 h of ischemia, the nNOS protein level determined by Western blots decreased slightly from the level of its normal control from the opposite limb. After 2 h of ischemia and 3 h of reperfusion, nNOS protein expression decreased to 63.9 ± 5.6% of normal, a significant difference from normal (P < 0.001) and the ischemia-only group (P < 0.05). eNOS protein expression decreased in response to both ischemia (80.9 ± 5.1%) and I/R (81.0 ± 4.2%). These changes were significant compared with their respective normal control (P < 0.01; Fig. 6). However, there was no band detected in any group to comigrate with iNOS positive control generated from macrophage.
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The immunohistochemical staining showed nNOS and eNOS in sections of the ischemia and I/R groups and no significant differences in the patterns of staining among the ischemia-only, I/R, and normal control groups. iNOS staining was not observed in any group.
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DISCUSSION |
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Using quantitative RT-PCR and Western blotting, we have demonstrated that ischemia and I/R differentially regulate the expressions of NOS mRNA and protein. After 2 h of ischemia, nNOS and eNOS protein expressions decreased. After 3 h of reperfusion, expression of nNOS and eNOS mRNA and protein decreased further. In contrast, iNOS mRNA significantly increased after ischemia and was further upregulated after I/R, while iNOS protein was not detected by either Western blotting or immunohistochemical staining.
No studies have examined altered expressions of NOS genes and proteins in the somatic nerve after I/R, although evidence suggests an important role for NOS in peripheral nerve injury. Our results support the hypothesis that NO may be involved in the mechanism of tissue injury including I/R injury (6). We have previously shown that supplementation of a NO donor significantly improves the microcirculation and contractile function of reperfused skeletal muscle (6, 25), whereas a NOS inhibitor exaggerates contractile dysfunction (18). That I/R induced significant reduction in the expression of eNOS mRNA and protein in the present study extends the findings of our earlier studies in skeletal muscle in which I/R downregulated eNOS mRNA to 28% of normal (34) and provides rationale for salvage NO therapy. The decrease in eNOS may play a role in exaggerating vessel spasm, neutrophil adhesion, and functional deficits in the early stages of reperfusion, thereby resulting in tissue injury and reperfusion failure. eNOS is mainly produced by endothelial cells and may be produced by Schwann cells (24, 27). Decreased eNOS mRNA and protein expressions may be linked to reperfusion-induced downregulation and/or reperfusion-induced cell damage with resultant failure to express eNOS mRNA.
The regulation of nNOS is exceedingly complex (38). Axotomy results in upregulation of nNOS mRNA in injured hypoglossal motoneurons (28) and increased nNOS immunoreactivity in pudendal motor neurons (33). However, the expression of nNOS mRNA in the present study was slightly downregulated after 2 h of ischemia and 3 h of reperfusion, and protein expression decreased after ischemia and even more after I/R. Evidently, the response to ischemia and I/R differs from that of hypoxia, which increased nNOS mRNA expression in nodose ganglion (32), although with the caveat that we only followed nNOS levels for 5 h. Although nNOS has been implicated in a wide variety of pathophysiological processes, the biological relevance of its decrease in I/R injury of the somatic nerve is unclear at this time, and further study is warranted.
Our data identify iNOS mRNA in normal somatic nerve of the rat, although expression is very low. The results extend our earlier findings in skeletal muscle (34) and, more generally, concur with results showing that the iNOS gene is expressed constitutively in airway epithelium, retina, cerebellum, and skeletal muscle tissue (4, 13, 28, 31, 33). iNOS is typically induced by cytokines and endotoxins and is thought to represent a physiological response of the immune system to trauma, infection, or tissue damage (31). Studies have shown that transcription and translation levels of iNOS were raised in rat sciatic nerve after a chronic constriction injury (23). Thus it is not surprising that iNOS mRNA was increased after ischemia and I/R in this study. iNOS is derived from macrophage, neutrophil, and Schwann cells. Thus we propose that iNOS mRNA during I/R may derive from both resident and infiltrating neutrophil and macrophage cells (19), as well as nerve cells (26). Two cytokines, tumor necrosis factor and interleukin-1, have been shown to be present in injured nerve (8, 29) and may be the source of induction.
Although iNOS mRNA was upregulated 14-fold from normal after 2 h of ischemia and 3 h of reperfusion, it was surprising to us that iNOS protein was not identified in either normal or treated nerve by Western blotting and immunohistochemistry. Several possible explanations should be considered. First, a time delay may be present between an increase in the rate of transcription and a concomitant change in the level of translation. Thus a corresponding increase in iNOS protein levels may occur on a longer time scale than our study. Second, posttranscriptional regulatory mechanisms to decrease protein expression or increase protein degradation may exist (1, 20). Third, it is conceivable that Western blotting and immunohistochemistry are simply not sensitive enough to detect iNOS protein in normal nerve or in tissue subjected to these experimental conditions. Nonetheless, we have little evidence for iNOS expression at the level of protein, suggesting a lesser role in either normal physiology or pathophysiology of early reperfusion of peripheral nerve.
Compared with our earlier results in skeletal muscle, ischemia and I/R resulted in an increased expression of iNOS mRNA, but the extent was much higher in the nerve (up to 14-fold) than in skeletal muscle (only 2.4-fold). The divergent results may be due to differences in the type of target tissue. A similar discrepancy exists in the literature: the effects of hypoxia on gene expression were not uniform among different cells and/or tissues (32, 36, 42).
In the early stage of NO research, it was assumed that the protective vs. destructive role of NO depended on concentration and isoform; that is, high NO concentrations produced by iNOS were implicated in damage to host cells (2, 9-11, 16, 26). Increasing evidence, however, suggests that NO may be beneficial or detrimental depending on the stage of evolution of the event (such as ischemia and I/R) and on the cellular compartment producing NO (11, 16), and injury (e.g., apoptotic mechanism) can be independent of NOS isoform or NO concentration. Our findings of the differential expression of distinct NOS mRNA and protein during ischemia and during early reperfusion in this study support this concept. Furthermore, the results of decreased eNOS mRNA and protein and increased iNOS mRNA in the I/R group in this study suggest the importance of determining the cellular and molecular mechanisms of transcription expressions of individual NOS.
In addition to three NOS mRNAs present in normal somatic nerve, we find nNOS protein in Schwann cells and axons. nNOS is thought to function as a neurotransmitter (2, 30) and may also be involved in the response to nerve injury (12, 38, 41) and regeneration (14, 15, 43, 44). It is difficult to understand, however, how NO, a short-lived molecule that is produced in the neuron cell body, can reach the distal axon. One alternative is that NO is locally synthesized. Indeed, our findings of nNOS in Schwann cells and axons appear to support this possibility. Schwann cells have an intimate relationship with axons and are involved in nutrition, impulse conduction, and nerve regeneration (11), perhaps by producing NO. The findings of nNOS-positive myelinated fibers (motor fibers) in this study contrast, however, with the data from normal spinal cord, where motor neurons are apparently nNOS negative (10). The precise mechanism of NO production and the exact role of NO in the function of somatic nerves, however, remain unknown.
In conclusion, the present study has demonstrated that three NOS genes (nNOS, iNOS, and eNOS) and nNOS and eNOS proteins are expressed in normal somatic nerve and are regulated by ischemia and I/R. The finding that I/R downregulates constitutive expression of NOS, especially eNOS, suggests a potential clinical benefit of NO donor in I/R injury.
Perspectives
Using a rat sciatic nerve I/R model in which the expression of mRNA and protein of three NOS isoforms was determined, we found that ischemia and reperfusion downregulated the expression of both mRNA and protein of nNOS and eNOS but upregulated iNOS mRNA. From a clinical perspective, future study should be directed toward determining dynamic expression of NOS isoforms; in addition, the microdomains in which individual NOS isoforms operate in I/R injury need to be more precisely characterized. An understanding of this modulation on a cellular and molecular level may lead to understanding the mechanisms of the reperfusion injury observed in vivo and to methods of ameliorating clinical outcome of peripheral nerve injury. ![]() |
ACKNOWLEDGEMENTS |
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The authors thank Laura P. Hale and Jie Li (Dept. of Pathology, Duke University Medical Center) for consulting and technical assistance with immunohistochemical evaluation.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-36046 (to J. R. Urbaniak) and HL-59130 (to J. S. Stamler).
Address for reprint requests and other correspondence: L.-E. Chen, Orthopaedic Microsurgery Laboratory, Box 3093, Duke University Medical Center, Durham, NC 27710 (E-mail: chen0006{at}mc.duke.edu).
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. Section 1734 solely to indicate this fact.
Received 6 February 2001; accepted in final form 11 April 2001.
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