1 Stokes Research Institute and Neonatology Division, Department of Pediatrics, Children's Hospital of Philadelphia, and 3 Department of Biochemistry and Biophysics and 2 Pulmonary and Critical Care Division, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104
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ABSTRACT |
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The present study identifies proteins
modified by nitration in the plasma of patients with ongoing acute
respiratory distress syndrome (ARDS). The proteins modified by
nitration in ARDS were revealed by microsequencing and specific
antibody detection to be ceruloplasmin, transferrin,
1-protease inhibitor,
1-antichymotrypsin, and
-chain fibrinogen. Exposure to nitrating agents did not deter the chymotrypsin-inhibiting activity of
1-antichymotrypsin. However, the ferroxidase activity of
ceruloplasmin and the elastase-inhibiting activity of
1-protease inhibitor were reduced to 50.3 ± 1.6 and 60.3 ± 5.3% of control after exposure to the nitrating agent. In
contrast, the rate of interaction of fibrinogen with thrombin was
increased to 193.4 ± 8.5% of the control value after exposure of
fibrinogen to nitration. Ferroxidase activity of ceruloplasmin and
elastase-inhibiting activity of the
1-protease inhibitor in the ARDS patients were significantly reduced (by 81 and 44%, respectively), whereas
1-antichymotrypsin activity was
not significantly altered. Posttranslational modifications of plasma
proteins mediated by nitrating agents may offer a biochemical
explanation for the reported diminished ferroxidase activity, elevated
levels of elastase, and fibrin deposits detected in patients with
ongoing ARDS.
nitric oxide; superoxide; ceruloplasmin; 1-protease
inhibitor; fibrinogen; oxidative stress
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INTRODUCTION |
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ACUTE RESPIRATORY DISTRESS SYNDROME (ARDS) describes an acute lung injury in which pathogenesis may be initiated by oxidative stress (21, 27, 35, 36). Oxidative stress is an imbalance created by either the overproduction of reactive species or the diminished rate of their elimination. In some pathological conditions such as hyperoxia, both overproduction and decreased detoxification of reactive species result in tissue injury (14, 27). The evidence for oxidative stress in ARDS originates from studies (5, 30) that show elevated levels of hydrogen peroxide in the exhaled air as well as in the urine of patients with acute lung injury. Increases in serum levels of lipofuscin and 4-hydroxy-2-nonenal, indexes of lipid peroxidation, have also been reported to correlate with the occurrence of ARDS in patients at risk for developing the disorder (37, 38), providing further evidence for oxidative stress-initiated injury. In addition to oxygen-derived reactive species, recent data indicated that nitrogen-derived species may also participate in pathological conditions. The contribution of nitrogen reactive species is determined by the potential protective effects of nitric oxide inhibitors, the use of nitric oxide knockout animals, and the presence of nitrated adducts, mostly nitrated proteins, localized at the site of injury (2, 3, 11, 12, 18, 29, 34).
Proteins are targets for reactive species, and the magnitude of protein modification correlated with the degree of oxidative and nitrative stresses (6, 11, 12, 15, 18, 34, 43). Posttranslational protein modifications induced by oxidative stress include carbonyls, which provide evidence for general oxidative modification of proteins (15, 43), and nitration of tyrosine residues to form 3-nitrotyrosine, which indicates the formation of nitrating species (20). Enzymatic [myeloperoxidase (MPO) (45) and eosinophil peroxidase (46)] and nonenzymatic sources (20, 33) can generate reactive nitrogen species capable of nitrating protein tyrosine residues in vitro. However, the proximal nitrating species in human disease have not been elucidated.
Recently, Lamb et al. (23, 24) reported that the levels of protein 3-nitrotyrosine, chlorotyrosine, and ortho-tyrosine are elevated in the bronchoalveolar lavage (BAL) fluid and plasma of ARDS patients. Other reports (8, 47) have also indicated an increase in nitric oxide production in acute lung injury and ARDS. Although these data support the hypothesis that oxidation and nitration reactions are indeed taking place, it provides limited information about which specific protein targets are modified by nitration. This study identified the prominent plasma proteins that are modified by tyrosine nitration in ARDS patients, supporting the existence of nitrative stress in ARDS. Nitration of proteins with or without concomitant oxidation affects the function of some of the proteins and thus provides a potential biochemical mechanism for the altered function of these proteins in ARDS.
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METHODS |
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Patient population and blood collection. All patients admitted to the Medical and Surgical Intensive Care Units at the Hospital of the University of Pennsylvania (Philadelphia, PA) during a 6-mo period were screened daily for ARDS by the same research coordinator. Patients were identified as having ARDS if they had an acute respiratory disorder, were intubated and receiving mechanical ventilation, and met the following criteria for ARDS proposed by the American-European Consensus Conference (7): 1) ratio of arterial PO2 to inspired oxygen fraction < 200; 2) bilateral lung infiltrates on chest radiograph consistent with pulmonary edema (specifically not due to atelectasis or pleural effusions); and 3) no clinical suspicion of left-sided congestive heart failure and, if measured, a pulmonary arterial capillary (occlusion) pressure < 18 mmHg. The day of onset was defined as the first day in which the patient met all the criteria within a 24-h period. Residual blood samples (collected during routine patient care in EDTA tubes) were identified daily and coded to protect patient identity and to eliminate bias in the performance of the biochemical analyses. The University of Pennsylvania Committee on Research on Human Subjects approved this study with a waiver of informed consent.
Twelve patients met the ARDS criteria during the period of screening and were used for analyses of nitrated proteins. The analyses for ARDS patients were performed with samples obtained on the third day after the onset of ARDS (2.9 ± 0.3 days). Plasma was separated from the cells, divided into aliquots, and frozen atProtein immunoprecipitation and gel electrophoresis. For immunoprecipitation of nitrated plasma proteins, each plasma protein sample (125 µg) was diluted in 500 µl of 20 mM Tris, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 4 mM EGTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 10 µg/ml aprotinin buffer, pH 7.4. For a positive control, plasma was incubated with 3-morpholinosydnonimine (SIN-1) at 1 mM final concentration for 1 h at 37°C. During the SIN-1 exposure, the protein solution was rigorously vortexed every 10 min to maintain the dissolved oxygen levels in the reaction mixture. This is a critical step because we found that the rapid consumption of oxygen will prevent the formation of superoxide and thus the formation of a nitrating agent. The samples were incubated at 4°C with shaking for 1 h with 15 µl of 20% (wt/vol) protein G-Sepharose beads. The supernatant was separated by centrifugation for 2 min at 10,000 rpm, and 2 µg of affinity-purified polyclonal anti-nitrotyrosine antibody were added to this supernatant and allowed to incubate overnight at 4°C with shaking. Samples were then incubated at 4°C for 1.5 h with 30 µl of protein G-Sepharose beads. The supernatant was removed after a 2-min centrifugation at 10,000 rpm, and the beads were washed three times with 500 µl of fresh buffer. After the final wash, the buffer was replaced with 50 µl of sample buffer and the samples were boiled for 5 min. The immunoprecipitated proteins were separated on a 10% SDS-PAGE and then were transferred to either nitrocellulose paper (NCP) or polyvinylidene difluoride membranes.
The NCP (Schleicher & Schuell, Keene, NH) was placed in a blocking solution of 10% dry milk powder in Tris-buffered saline (TBS) at pH 7.5 for 1 h, followed by three 5-min washes in TBS-0.1% Tween (TBS-T). The blot was then incubated for 2 h with monoclonal anti-nitrotyrosine antibody diluted to 1:1,000 in the TBS-T with 1% dry milk powder antibody buffer. After being washed with two 5-min washes of TBS-T, the blot was incubated for 1 h with anti-mouse IgG diluted 1:2,000 in antibody buffer. Blots were then washed extensively with four 5-min washes in TBS-T and two 5-min washes in TBS and developed with the enhanced chemiluminescence Western blotting system (Amersham Pharmacia Biotech). Blots transferred to polyvinylidene difluoride membrane were stained with Amido Black protein stain, and selected bands were excised for sequencing by being subjected to conventional Edman degradation at the Protein Core Facility at the Wistar Institute (Philadelphia, PA). Specific antibodies against human proteins were obtained from DAKO (Carpenteria, CA), and antibodies to transferrin were a gift from Dr. Pinchas Cohen (Children's Hospital of Philadelphia, Philadelphia, PA).In vitro exposure of proteins and determination of specific
activities.
Human plasma proteins (Calbiochem, San Diego, CA) were dissolved (5.0 mg/ml) in 100 mM potassium phosphate-25 mM sodium bicarbonate buffer,
pH 7.4, with 0.1 mM diethylenetriaminepentaacetic acid (DTPA). To
nitrate tyrosine residues in proteins, we utilized a bolus addition of
peroxynitrite in the presence of CO2 (generated by the
added bicarbonate in an open system). The yield of nitration was
measured by isotope dilution by gas chromatography-mass spectroscopy (GC-MS; Hewlett-Packard) as previously described (34). The protein samples were hydrolyzed in 6 N HCl containing 1% phenol, and 10 nmol
of the isotopically labeled internal standard
3-nitro[15N]tyrosine were added to the
hydrolysates. Ceruloplasmin ferroxidase activity was measured with
o-dianisidine dihydrochloride as substrate at 30°C (17).
Transferrin and apo-transferrin assays measured the release of iron or
iron-binding activity as described in a prior study (26). The
activities of 1-antichymotrypsin and
1-protease inhibitor were measured by the inhibition of
chymotrypsin and elastase activity, respectively (10). The activity of
fibrinogen was measured by the increase in turbidity of a thrombin
solution. Untreated or treated fibrinogen (0.5 mg/ml) was incubated
with 0.024 U/ml of thrombin at 37°C in 50 mM potassium phosphate
buffer, pH 7.4, and the change in absorbance at 360 nm was measured for 1 h (41). The protein concentration in the plasma of control subjects
and ARDS patients was determined by a solid-phase ELISA (6). Briefly,
serial dilutions of plasma proteins and purified plasma proteins were
immobilized on NCP and probed with anti-human antibodies conjugated
with horseradish peroxidase (1:5,000 dilution). The NCP membrane was
then developed with enhanced chemifluorescence reagent (Amersham
Pharmacia Biotech). The chemifluorescence of each sample was measured
by scanning the blot with a STORM 840 imaging detector. The
fluorescence was measured as net counts corrected for background from a
sample blank with ImageQuant analysis software and then plotted on a
semilogarithmic plot. The concentration of the protein in each sample
was determined from the linear portion of the sigmoidal curve from the
semilogarithmic plot of net counts versus the antigen concentration of
the purified protein standard. The following values were obtained in
the healthy individuals: 14.2-51.5 mg/dl ceruloplasmin, 56-62
mg/dl
1-antichymotrypsin, and 106-329 mg/dl
1-protease inhibitor. These values compare well with the
values reported by Burthis and Ashwood (9): 18-45 mg/dl for
ceruloplasmin, 30-60 mg/dl for
1-antichymotrypsin, and 78-200 mg/dl for
1-protease inhibitor.
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RESULTS |
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Immunoprecipitation of nitrated proteins from ARDS patients and
control subjects.
Plasma proteins modified by nitration were immunoprecipitated with
affinity-purified polyclonal anti-nitrotyrosine antibodies. Figures
1 and 2, lane 1, shows the presence
of distinct protein bands modified by nitration in ARDS patients, which
are not evident in healthy subjects. Plasma from healthy
individuals incubated at 37°C with SIN-1, which cogenerates nitric
oxide and superoxide and nitrates plasma proteins (16, 33), was used as
a positive control. The apparent molecular masses of the proteins
modified by nitration were 120, 73.6, 64.9, 58.1, and 56 kDa.
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Identification of the nitrated proteins.
A representative blot of immunoprecipitated plasma protein stained with
Amido Black is shown in Fig. 2, lane
2. Bands of nitrated proteins with equal molecular masses were
pooled from six different patients and subjected to sequencing.
Microsequencing identified the protein with an apparent molecular mass
of 56 kDa to be the -chain of fibrinogen. Because of the high
molecular mass, the concentration of the other bands obtained by
immunoprecipitation was not sufficient for sequencing. To reveal the
identity of the other proteins, we employed specific anti-human
antibodies that have been previously utilized for the quantification of
plasma proteins (9). Immunoblotting with different anti-human
antibodies to plasma proteins after immunoprecipitation of nitrated
proteins revealed the following: the 58.1-kDa band was detected by the
1-protease inhibitor antibody, the 64.9-kDa band by the
1-antichymotrypsin antibody, the 73.6-kDa band by the
transferrin antibody, and the 120-kDa band by the ceruloplasmin
antibody (Fig. 2). Immunoblotting with anti-plasminogen antibody did
not reveal the presence of plasminogen among the immunoprecipited
nitrated proteins (Fig. 2). Similarly, no binding was present in the
immunoprecipitated bands with antibodies against ferritin and albumin
(data not shown). Immunoblotting with the anti-human antibodies to
plasma proteins after immunoprecipitation of the proteins from healthy
subjects did not reveal any positive binding (Fig. 2). To ascertain
that plasminogen was in fact present but simply not nitrated, the same set of antibodies was used to visualize these proteins on Western blots
(Fig. 3).
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Protein function after exposure to peroxynitrite-CO2 in
vitro.
The proteins were exposed to peroxynitrite-CO2, the extent
of nitration was evaluated by GC-MS (34), and the effect
of the exposure to the activity of the protein was determined (Table 1). Exposure of ceruloplasmin
resulted in nitration of nearly three tyrosine residues per molecule
and a 50% decrease in ferroxidase activity. Similarly the
anti-elastase activity of 1-protease inhibitor was
reduced by 40% in the exposed protein. However, the activity of
1-antichymotrypsin was not altered on exposure of the
protein to peroxynitrite-CO2 and nitration of nearly two tyrosine residues per protein molecule (Table 1). Exposure of iron-loaded transferrin to peroxynitrite-CO2 did not result
in the release of iron, and nitration of apo-transferrin only
marginally affected iron binding (data not shown). This confirmed the
results of a previous study (26) that had shown that nitration of
iron-loaded transferrin did not induce the release of iron and that
nitration of apo-transferrin only marginally inhibited the binding of
iron to the protein. The iron-binding activity of the apoprotein is significantly inhibited only after extensive nitration (8 residues/mol of protein) of the protein (26). Exposure of fibrinogen to
peroxynitrite-CO2 resulted in the acceleration of
thrombin-catalyzed clot formation and the nitration of one tyrosine
residue per fibrinogen molecule (Table 1).
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Specific enzymatic activities in ARDS patients.
Table 2 describes the specific activities
of ceruloplasmin, 1-protease inhibitor, and
1-antichymotrypsin in control subjects and
ARDS patients. There was significant reduction by 81% of the ceruloplasmin ferroxidase activity despite a 2.4-fold increase in
the plasma protein level of ceruloplasmin [0.30 ± 0.08 (SE) vs.
0.71 ± 0.04 µg/ml; P < 0.05]. The specific
anti-elastase activity of
1-protease inhibitor was also
significantly reduced by 44%. The plasma protein concentration of
1-protease inhibitor was increased twofold in the ARDS
patients (1.82 ± 0.51 vs. 3.85 ± 0.27; P < 0.05).
Consistent with the in vitro data, the specific activity of
1-antichymotrypsin was not significantly reduced in the
ARDS patients. The concentration of
1-chymotrypsin
inhibitor was increased 3.4-fold in the plasma of ARDS patients (0.88 ± 0.30 vs. 2.96 ± 0.10, P < 0.05).
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DISCUSSION |
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Protein oxidation and nitration have previously been utilized as
indirect indexes of oxidative stress in human and animal models of
disease (2, 3, 11, 12, 15, 18, 29, 34, 43). However, with the exception
of one study (29), the specific protein(s) modified by nitration in
human disease is not known. This study identified the specific proteins
that are nitrated in patients with ARDS. Consistent with previous
observations (2, 16, 29), only a few plasma proteins are modified by
nitration in ARDS patients. The predominant plasma proteins modified by nitration in ARDS patients, listed in order of descending molecular mass, are ceruloplasmin, transferrin 1-antichymotrypsin,
1-protease inhibitor, and
-chain fibrinogen and
appear to fall into three major categories: proteins that regulate
metal binding, proteolysis, and coagulation. The activities of these
proteins are critical for maintaining normal homeostasis and protecting
the lung from oxidative injury. Previous data indicated that these
pathways are altered in patients with ongoing ARDS. The BAL fluid
levels of ceruloplasmin increase in ARDS patients, possibly as a
response to a greater need for antioxidant activity in the lung (17, 22, 28). Despite the increase in BAL fluid levels, the ferroxidase activity of ceruloplasmin is significantly diminished in patients with
acute lung injury, suggesting that the activity of the protein is
impaired (17). The
1-protease inhibitor and the
1-anti-chymoptrypsin are acute-phase reactants, with
their levels rising in response to stress to inhibit the action of
proteolytic pathways (trypsin, elastase, and chymotrypsin) and thus
maintain normal protein turnover and structural integrity of the lower
respiratory tract. The rise in the levels of these proteins in plasma
is evident in the ARDS patients (Fig. 3), particularly for
1-antichymotrypsin, which is barely detected in healthy
individuals and is increased 3.4-fold in ARDS patients. However,
despite the rise in protein levels of these inhibitors, patients with
acute lung injury have been shown to have high elastase activity in BAL
fluid, and the concentration of elastase correlated with the severity
of injury (10, 25, 44). Finally, previous studies (4, 19, 39) indicated that patients with acute lung injury have fibrin deposits in their alveoli and capillaries as a likely consequence of the increase in
procoagulants and the decrease in fibrinolytic activities. Overall, the
function of proteins found to be modified by nitration appears to be
altered in the plasma of patients with ongoing ARDS.
Nitration of tyrosine residues in these proteins may not be the single biochemical mechanism responsible for the alteration of function because oxidation of other critical amino acids such as methionine, cysteine, and tryptophan also contributes to the alteration in function. This is likely in view of the observations that oxidants and nitrating agents are generated during acute lung injury (5, 23, 24, 27, 30, 37, 38) and some of the proteins are exquisitely sensitive to both oxidation and nitration. Moreover, most known nitrating agents are also capable of oxidizing tyrosine, cysteine, methionine, and tryptophan residues in proteins (1, 20, 34, 46, 47). However, both protein oxidation and nitration appear to be selective (2, 16, 29, 40, 43). The proteins modified by oxidation after exposure of human plasma to oxidants was independent of the abundance of the protein because albumin was modified to a lesser extent than less abundant proteins such as transferrin and fibrinogen (40). Recent data (42) revealed that, similar to oxidation, the selectivity of protein nitration is not a function of the abundance of protein, the number of tyrosine residues, or the nature of the nitrating agent. The local environment of relatively surface-exposed tyrosine residues appears to determine the sites of nitration. Several nitrating agents with high efficiency nitrate the same tyrosine residue in proteins (42). Moreover, other data indicated that nitration of proteins appears to be favored over nitration of free tyrosine after exposure of human plasma to different nitrating agents (33).
Exposure of plasma proteins to nitrating agents in vitro resulted in
the inhibition of function (1-protease inhibitor,
ceruloplasmin), a gain of function (fibrinogen), or no effect
(
1-antichymotrypsin, transferrin). Both ceruloplasmin
and
1-protease inhibitor are sensitive to oxidation,
and, therefore, it is likely that oxidation and nitration may
contribute to the loss of function (32, 40). Nitration of
1-protease inhibitor has been shown to inactivate the
inhibitory function of this protein toward elastase but not toward
trypsin and chymotrypsin inhibitory activity (13, 31). However,
exposure of
1-protease inhibitor to peroxynitrite in the
absence of CO2 was also shown to inactivate the elastase
activity by oxidation of methionine residue(s) (32). In contrast to the loss of elastase activity, the function of
1-antichymotrypsin is not inhibited by exposure to
nitrating agents (Table 1) and the
1-antichymotrypsin
activity in the plasma of patients with ARDS did not significantly vary
from that in control subjects (Table 2). This may be important for the
rate of clearance of nitrated proteins from the plasma. Chymotrypsin
was found to be capable of cleaving next to nitrated tyrosine residues
but at a considerably slower rate than tyrosine residues (Souza JM,
Choi I, Chen Q, Daikhin E, Yudkoff M, Obin M, Ara J, Horwitz J, and Ischiropoulos H, unpublished results). The slower rate of
degradation together with the presence of increased levels of
1-antichymotrypsin may extend the circulating half-life
of nitrated proteins in the plasma.
The most unexpected finding was that the exposure of fibrinogen to
nitrating agents results in the acceleration of the interaction with
thrombin and clot formation (Table 1). In contrast to the effect of the
nitrating agents, previous observations (41) indicated that oxidation
of fibrinogen results in a dramatic loss of thrombin-catalyzed clot
formation. Although we cannot exclude the possibility that the
nitrating agents oxidized residues different from those oxidized by
radiation or exposure to iron ascorbate (41), nitration of fibrinogen
may be responsible for the increased clot formation. Plasminogen, which
is critical for fibrinolysis, was not nitrated in the plasma of ARDS
patients (Fig. 2) and presumably maintains its catalytic activity.
Plasminogen is a 95- to 97-kDa protein with 3.7 mol% tyrosine and
contains 24 disulfide bridges (9). Putative in vivo nitrating agents
such as the peroxynitrite-CO2 adduct, SIN-1 in the presence
of CO2, and
MPO-H2O2-NO2 nitrate tyrosine residues that are exposed to the surface of the protein in a local environment free of disulfide bridges or other steric hindrances (42). The presence of 24 disulfide bridges in
plasminogen is likely to provide sufficient steric hindrance, preventing tyrosine residue nitration. Therefore, the presence of
fibrin deposits in ARDS patients (4, 39) may be the result of
accelerated clot formation due to alterations in the activity of fibrinogen.
Overall, the presence of nitrated proteins in ARDS indicates the generation of nitrating agents and suggests the existence of oxidative and nitrative stress during the development of the disease.
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ACKNOWLEDGEMENTS |
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We thank Drs. M. Beers, A. Fisher, and S. Thom (University of Pennsylvania, Philadelphia, PA) for critical reading of the manuscript, D. Reim (Wistar Institute, Philadelphia, PA) for sequencing, and M. Whiteman (King's College London, London, UK) for suggestions with the elastase assay.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute (NHLBI) Grant HL-54926; NHLBI Specialized Center of Research In Acute Lung Injury Grant P50-HL-60290 (to H. Ischiropoulos and P. N. Lanken); and National Institute on Aging Grant AG-16987 (to H. Ischiropoulos).
R. F. Foust was supported by National Heart, Lung, and Blood Institute National Research Service Award HL-07748. H. Ischiropoulos is an Established Investigator of the American Heart Association.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: H. Ischiropoulos, Stokes Research Institute, Children's Hospital of Philadelphia, 416D Abramson Center, 34th St. and Civic Center Blvd., Philadelphia, PA 19104-4318 (E-mail: ischirop{at}mail.med.upenn.edu).
Received 10 August 1999; accepted in final form 17 December 1999.
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