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Abstract |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Air breathing, especially oxygen therapy, exposes the lung to reactive oxygen species (ROS). Antioxidant enzymes (AOEs) may protect the lung from ROS-mediated injury. Because expression of the key AOEs increases in several animal species during gestation, we investigated (1) the messenger RNA (mRNA) and activity levels of the key AOEs manganese and copper-zinc superoxide dismutases (MnSOD and CuZnSOD, respectively), catalase (CAT), and glutathione peroxidase (GPx) in adult lung samples and during ontogenesis; and (2) the difference in AOE expression between lung and liver. In the lung, the mRNA expression of MnSOD, CuZnSOD, and CAT increased toward adulthood, and GPx was unchanged. Pulmonary activities of MnSOD and CuZnSOD were unchanged, whereas CAT increased 3-fold from fetuses to adults. In the liver, the mRNA expression of MnSOD, CuZnSOD, and GPx increased, whereas that of CAT decreased toward adulthood. Hepatic activities of MnSOD and CuZnSOD increased 2-fold and 4-fold, respectively, whereas CAT was similar in fetuses and adults. Neonatal GPx activity was 2-fold higher in the lung and 6-fold higher in the liver compared with adults. The mRNA levels of MnSOD correlated positively with those of CuZnSOD and CAT in the lung, and GPx with those of MnSOD and CuZnSOD in the liver. Activities of MnSOD and CuZnSOD correlated positively in the liver. We conclude that the regulation of AOEs differs between human lung and liver, and is not tightly coordinated in either tissue.
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Introduction |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Postnatally through adult life, pulmonary tissue is exposed to higher concentrations of oxygen than other organs, which renders it vulnerable to free radical-mediated injury (1). Furthermore, the production of reactive oxygen species (ROS) by lung cells is increased if these cells are exposed to hyperoxia (4, 5). Pulmonary defense mechanisms against ROS-mediated injury include various small molecular-weight antioxidants and antioxidant enzymes (AOEs). In aerobic organisms the most important AOEs are manganese and copper-zinc superoxide dismutases (MnSOD and CuZnSOD, respectively), catalase (CAT), and glutathione peroxidase (GPx) (6, 7). Although the expression and developmental profile of AOEs in mammalian tissues have been explored in a number of studies, very little is known about these enzymes in human tissues. Since the expression of AOEs appears to be species-specific, the regulation of these enzymes in human tissues is important for understanding the pathogenesis of various lung diseases in both newborn infants and adults.
The regulation of pulmonary AOEs in response to hyperoxia is complex, and both age- and species-dependent
(8). Exposure of adult animals to 95% oxygen is lethal
with no AOE response, whereas neonates of many animal
species survive and show hyperoxia-induced increases in
lung AOE expression (10, 11). Furthermore, the regulation of different AOEs is not uniform. Exposure of adult rats to 85 to 95% oxygen results in increased messenger
RNA (mRNA) expression of MnSOD (14, 15) and GPx
(15), whereas CAT and CuZnSOD mRNA expression remain unchanged (12, 14, 15). Simultaneously, the specific
activities of these enzymes have been found to increase individually in some (16), but not all (14, 15), studies. Resistance to hyperoxia can be achieved by treating adult
rats with tumor necrosis factor (TNF)-
(19, 20), with endotoxin (14, 21, 22), or by repeated exposures to hyperoxia
(8, 23), and it is associated with simultaneous induction
of AOEs (8, 14, 19, 24, 25). On the other hand, increased resistance to hyperoxia has also been reported
without induction of any AOEs (26).
The expression of AOEs in fetal lung of various mammals increases toward term (13, 27). This has been considered a defense mechanism against air breathing and relative hyperoxia after birth. Newborn preterm infants suffering from respiratory failure are exposed not only to relative hyperoxia in the extrauterine environment but also to mechanical ventilation with high inspired oxygen levels and high airway pressures, both of which are considered risk factors for bronchopulmonary dysplasia (33, 34). Thereby, insufficient activities of AOEs in the lungs of preterm infants may further aggravate the damage resulting from the unavoidable ventilatory care.
Some investigators have measured individual AOE activities in adult human lung (9, 35). Despite the obvious ethical and practical difficulties in obtaining tissue samples, some studies to elucidate the ontogenesis of some of these enzymes have also been reported (9, 35). However, we are not aware of a systematic investigation of the expression patterns of the most important AOEs in human lung. The aims of the present study were therefore (1) to measure the mRNA levels and activities of MnSOD, CuZnSOD, CAT, and the classical form of GPx in adult human lung samples and during ontogenesis; and (2) to compare the developmental pattern of AOE expression in human lung with that in human liver.
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Materials and Methods |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Tissue Samples
Fetal lung and liver samples were obtained from six legal abortions (15 to 19 gestational wk) (Nos. 1-6). The delay from delivery room to laboratory was approximately 1 h. The samples between 35 and 42 gestational wk were obtained from three neonatal autopsies (Nos. 7-9) performed within 12 h of death. The causes of death were congenital heart disease (No. 7), respiratory failure and hydronephrosis (No. 8), and birth asphyxia with meconium aspiration (No. 9). Adult lung tissue samples were obtained from macroscopically normal tissues of lung cancer patients undergoing lung surgery (Nos. 10-14), and from donor lungs of single-lung transplantations (Nos. 15 and 16). Three of the adult samples represent lung biopsies from patients with 10 to 30 yr smoking history (Nos. 10-12), and two are nonsmokers with completely normal lung histology (Nos. 13 and 14). Healthy adult liver tissue was obtained from partial liver transplantations (Nos. 10- 12). For adult tissues, the delay from operating room to laboratory was less than 30 min.
The lung and liver tissues were frozen in liquid nitrogen
and stored at 
80°C. The study protocol and the sampling
procedures were approved by the Ethical Committees of
the Hospital for Children and Adolescents and the Department of Thoracic and Cardiovascular Surgery, University of Helsinki, Helsinki, Finland.
RNA Analysis
Total RNA was extracted from the tissues using the acid-
guanidium method (39) and fractionated on denaturing
agarose gels at 10 µg/lane (15 µg/lane for MnSOD). Following capillary transfer onto nylon filters (Hybond-N;
Amersham International, Amersham, UK), the blots were
hybridized with 32P-labeled (DuPont, Zaventem, Belgium)
complementary RNA probes using standard methods (40).
As templates for transcription, we used complementary
DNA (cDNA) clones representing nucleotides 596 to 987 of human MnSOD (41), 127 to 457 of human CuZnSOD (42), 537 to 2218 of human CAT (43), and 533 to 624 of rat GPx (44) cloned into the pSP65 vector (Promega Co.,
Southampton, UK). The 91-base pair cDNA probe for rat
GPx1 is highly homologous to human GPx1 cDNA. The
cDNAs were kindly provided by Dr. Y.-S. Ho (Wayne
State University, Detroit, MI). Following hybridization and washes, the filters were exposed to Kodak BioMax
MR autoradiography film (Eastman Kodak Co., Rochester, NY) at 80°C for 12 h to 7 d. The filters were stored
for 3 to 22 mo at 4°C, and then rehybridized with 
-actin
and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) control probes transcribed from p-TRI--actin plasmid
and p-TRI-GAPDH-plasmid, respectively (Ambion, Austin, TX). The mRNA levels were quantified relative to the
-actin and GAPDH signals by X-Rite 331 Transmission densitometer (X-Rite, Grandville, MI).
Biochemical Studies
For enzyme activity assays, 100 mg of thawed tissue were extensively homogenized in ice-cold 1% Triton X-100 in 0.01 M phosphate-buffered saline solution, pH 7.2, and then filtered through a double-layered cheesecloth.
The xanthine oxidase-cytochrome c method was used to assay total SOD activity (CuZnSOD and MnSOD), and MnSOD activity was distinguished from CuZnSOD activity by its resistance to 1 mM potassium cyanide (45). CAT activity was determined with an oxygen electrode as previously described (46), and GPx activity by measuring the oxidation rate of reduced nicotinamide adenine dinucleotide phosphate in the presence of t-butylhydroperoxide, glutathione, and glutathione reductase (47).
Enzyme activities are expressed as units per milligram of protein and units per milligram of DNA to compensate for the possible effect of blood remaining in the tissues and to take into account the change in the protein/DNA ratio that occurs during lung development (48, 49). Protein and DNA were measured by the methods of Lowry and colleagues (50) and Vytásek (51), respectively.
Statistical Analysis
For statistical analysis of results, Mann-Whitney U test
was used with a level of P 
0.05 chosen to indicate significant differences. Adult samples (lung n = 7, liver n = 3)
were compared with fetal (lung and liver n = 6) and neonatal samples (lung and liver n = 3). Spearman rank correlation coefficient was used to assess the correlation between enzyme activity and mRNA levels.
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Results |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Lung
MnSOD. Human MnSOD is transcribed as 4-kb and 1-kb mRNA species (52, 53). Both transcripts were expressed in adult and neonatal lung, but in fetal lung the 1-kb species was undetectable (data not shown). The levels of the 4-kb transcript in the adult lung samples were variable (Figure 1A). They were high in two longtime smokers (lanes 10 and 11) and in donor lungs of single lung transplants (lanes 15 and 16), and lowest in two nonsmokers with normal lung histology (lanes 13 and 14). The highest level of the 4-kb transcript was found in a neonatal patient (lane 9), who died of meconium aspiration after 18 h ventilation with 70 to 100% oxygen. The other two neonatal samples had low mRNA expression, but the fetal samples had even lower expression (Figure 1A). MnSOD activities in the adult lung samples were similar to those in the neonatal and fetal samples when standardized against protein or DNA (Figure 2A). One fetal sample had no detectable activity. There was no correlation between the 4-kb mRNA levels and the specific activities expressed as units per milligram protein, whereas activities expressed as units per milligram DNA correlated with the 4-kb mRNA levels (r = 0.57, P = 0.03).
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, median. Fetal samples were obtained from
legal abortions, neonatal samples from autopsies, and adult samples from lung donors of single lung transplantations and surgical
resections.
CuZnSOD. CuZnSOD is also transcribed as two mRNA species, 0.9 kb and 0.7 kb in length (54). Both were expressed in the adult lung samples, in which the 0.7-kb species was three to four times more abundant than the 0.9-kb species (Figure 1B). In the neonatal samples, both mRNA species were expressed to approximately the same extent as in the adult samples, whereas in the fetal samples, with one exception, the levels were lower than in the neonatal and adult samples (Figure 1B). Although there was considerable variation in the level of expression of both transcripts, they generally varied in parallel (r = 0.93, P < 0.001).
CuZnSOD activity in neonatal lung was lower (P = 0.02) than in fetal lung when expressed as units per milligram protein but the difference in the activity expressed as units per milligram DNA was not significant. The activities in the adult samples did not differ from those in the neonatal or fetal samples (Figure 2B). Neither of the CuZnSOD mRNA species correlated with the activities of the same samples when standardized against protein or DNA.CAT. The expression of CAT mRNA was high and somewhat variable in adult lung. There was a clear developmental trend, in that the neonatal lung had approximately one-half the adult mRNA levels, and the fetal lung even lower (Figure 1C).
CAT activity, standardized either against protein or DNA, was lower in fetal than in neonatal (P = 0.02) or adult (P = 0.003) lung (Figure 2C). In addition, CAT activity expressed either as units per milligram protein (r = 0.69, P = 0.008) or units per milligram DNA (r = 0.60, P = 0.02) correlated positively with the mRNA levels of the same samples.GPx. GPx differed from the other three AOEs in that fetal, neonatal, and adult lung had similar levels of mRNA expression (Figure 1D).
GPx activity standardized against DNA was lower (P = 0.04) in the fetuses than in the neonates, but the difference between the groups was not significant when standardized against protein (Figure 2D). On the other hand, the adults had lower (P = 0.03) GPx activity level (milliunits per milligram protein) than the neonates (Figure 2D). GPx activity standardized against protein or DNA did not correlate with the mRNA pattern of the same samples. The MnSOD 4-kb transcript correlated positively with 0.7-kb (r = 0.76, P = 0.003) and 0.9-kb (r = 0.80, P = 0.002) transcripts of CuZnSOD, 2.4-kb transcript of CAT (r = 0.76, P = 0.003), and 1.1-kb transcript of GPx (r = 0.62, P = 0.02). Because the mRNA levels standardized against-actin signal varied substantially, the AOE mRNA
levels were also quantified relative to GAPDH signal, but
the pattern of expression was similar (Figure 1). Of these four enzymes, only CAT and GPx activities expressed as
units per milligram DNA correlated with each other (r = 0.65, P = 0.01) in the lung.
Liver
MnSOD. Both the 4-kb and 1-kb transcripts of MnSOD were detectable in the adult and neonatal liver samples, whereas in the fetal samples the 1-kb species was undetectable (data not shown). The expression of the 4-kb species increased toward term and adulthood (Figure 3A). The neonates and adults had approximately similar levels of mRNA, although considerable variation, especially between the adults, was observed (Figure 3A).
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CuZnSOD. The expression of both CuZnSOD mRNA transcripts was similar during the early fetal period and at term, but especially the 0.7-kb transcript increased toward adulthood (Figure 3B). As in the lung, the 0.7-kb and the 0.9-kb species varied in parallel (r = 0.96, P = 0.003).
CuZnSOD activity was 4-fold higher (P = 0.02) in the adults than in the fetuses. Furthermore, adults had higher (P < 0.05) levels of activity (units per milligram protein) than did the neonates (Figure 4B). There was, however, no difference when activity was expressed as units per milligram DNA.CAT. CAT mRNA declined after birth (Figure 3C).
CAT activity did not differ between the age groups when standardized against protein (Figure 4C). The activity, standardized against DNA, was higher in the neonates than in the fetuses (P = 0.02) or adults (P < 0.05).GPx. GPx mRNA expression increased postnatally (Figure 3D).
GPx activity, expressed as milliunits per milligram protein, did not change significantly when fetal activity levels were compared with neonatal and adult levels (Figure 4D). The adults, however, had lower (P < 0.05) levels of activity (milliunits per milligram protein) than did the neonates. Activity standardized against DNA was higher in the neonates than in the fetuses (P = 0.02) or adults (P < 0.05). When the specific activities of these four enzymes in the liver were correlated with the corresponding mRNA levels of the same samples, only MnSOD activity expressed as units per milligram protein showed a significant positive correlation with the 4-kb mRNA species (r = 0.85, P = 0.008). In the liver, the 1.1-kb species of GPx correlated with the 4-kb species of MnSOD (r = 0.67, P = 0.03) and with the 0.7-kb (r = 0.86, P = 0.007) and 0.9-kb (r = 0.73, P = 0.02) species of CuZnSOD. The mRNA levels were quantified relative to both-actin and GAPDH signals,
but the results were similar (data not shown). MnSOD activities standardized against protein (r = 0.75, P = 0.02)
and DNA (r = 0.86, P = 0.006) correlated with CuZnSOD
activities of the same samples.
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Discussion |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Although a variety of mechanisms contribute to protection against ROS-mediated cell and tissue injury, intracellular AOEs are considered to play a major role. MnSOD
has been suggested to be the key AOE responsible for the
protection of the lung against hyperoxia-induced injury
(14, 55, 56). MnSOD is induced by hyperoxia (15, 16, 18)
and by cytokines such as TNF- (57, 58), interferon-
(59),
interleukin (IL)-1 (60), and IL-6 (63). The developmental regulation of MnSOD appears to be species-specific; for example, in guinea pig lungs MnSOD mRNA
(32) increased toward term, whereas in rat lungs neither mRNA nor activity showed such a trend (30). On the
other hand, MnSOD protein concentration in rat lung increased toward term (29). Few studies on the expression of
MnSOD in human tissues are available. In human bronchial epithelium in vivo, MnSOD mRNA was not upregulated during a 12-h exposure to 100% oxygen (64). In an
immunohistochemical study on human lung, MnSOD protein increased in the peripheral airways during development (65).
In this study, the mRNA of MnSOD was lower in the
fetuses than in the adults. Large variation in the mRNA
levels between the various individuals was observed. Any
study of humans encounters problems of obtaining adequate tissue samples. After induced abortion and neonatal
death, there is a variable time before autopsy can be performed. The clinical disorder and its treatment may also influence the variables under study, for example, through
enzyme induction. Even though typical respiratory distress
syndrome cases had been excluded, the term infants had
been treated in the intensive care unit for 18 h to 5 d. This
is ample time for an increase of inflammatory mediators,
such as TNF-, and for an influx of neutrophils into the
lung, which may account for MnSOD induction. In addition, one of the lung donors had suffered from multiple trauma, which may have resulted in cytokine-mediated
MnSOD induction. Thus, a major source of variation of
our results may be altered gene expression superimposed
upon ontogenesis. In this study, the mRNA of MnSOD did
not correlate with the enzyme activity expressed as units
per milligram protein in the lung. This finding is in agreement with previous studies, which have demonstrated that hyperoxia-induced increase in MnSOD mRNA expression
did not correlate with the specific enzyme activity in rat
lung, and that a large portion of the MnSOD protein was
enzymatically inactive (15, 56). Our results suggest that
MnSOD mRNA can be upregulated in adult and neonatal
human lung and liver. Because MnSOD activity was higher
in the liver than in the lung, oxygen itself may not be the
only factor resulting in upregulation of this enzyme. Alternatively, MnSOD may be induced only in a subpopulation
of lung cells.
In contrast to MnSOD, in most studies CuZnSOD is not upregulated by cytokines (57, 61, 63) and is induced by hyperoxia to a lesser degree than MnSOD or not at all (15, 56). Rabbit lung total SOD activity (27) and rat lung CuZnSOD activity and mRNA (28, 30) increased toward term and adulthood. In human lung, however, CuZnSOD activity did not change toward birth (35, 37), but contradictory results have also been reported (9). Our results showed that CuZnSOD mRNA expression increased in the lung and liver, and that the enzyme activity increased only in the liver during development. The 0.7-kb mRNA species of CuZnSOD was two to three times more abundant than the 0.9-kb species in both tissues, which is in agreement with previous results on human cell lines (54). The differences between the various individuals were smaller than those of MnSOD, possibly due to the minimal effect of inflammatory cytokines on CuZnSOD.
The induction and importance of CAT in human lung is poorly defined. In animals and cell culture models, CAT was induced by hyperoxia, oxidants, and cytokines in some (19, 20, 66, 67) but not all (15, 68) studies. CAT mRNA and activity levels increased during development in rabbit, rat, and guinea pig lung and liver (27, 30, 32, 69). In one study, CAT was not upregulated following 12 h exposure to 100% oxygen in human bronchial epithelial cells (64). CAT activity increased prenatally in human lung and postnatally in human liver (35), but the developmental profile of CAT mRNA expression in human tissues has not been defined. Our results showed that CAT was the only AOE to increase in both the activity and mRNA expression throughout development in the lung but not in the liver, a fact that raises a question of the role this enzyme may have in pulmonary defense.
Intracellular (the classical form) and extracellular forms of GPx have been described (70). In previous studies, the mRNA expression and activity levels of intracellular GPx were upregulated to some extent by hyperoxia and cytokines (15, 19, 20, 71). GPx activity and mRNA expression increased in rat, mouse, and guinea pig lung in late gestation (30, 32, 72). Two previous studies on GPx activity in human lung reported large interindividual variation, but no developmental trend was observed (35, 36). In our study, GPx mRNA expression in the lung was constant during the whole period studied, and in agreement with previous animal studies (48, 72), GPx activity declined postnatally toward adulthood both in the lung and in the liver.
Because -actin expression can vary during development, we used both -actin and GAPDH as the reference
in Northern blots. The mRNA expression using GAPDH
as the reference was similar to that with -actin for all enzymes studied both in the lung and in the liver. Therefore,
it follows that possible variations in -actin expression
during development do not explain the lack of correlation
of the mRNA levels with the activity of the same enzyme.
A discordance between the AOEs mRNA levels and specific activity has been previously reported in animal studies (32, 73), and it appears that additional post-transcriptional regulation mechanisms may be involved in AOE
gene expression at different periods of development in human tissues as well.
Activity measurements of all the enzymes were standardized against cell protein and DNA. The former may underestimate the real activity levels, if pulmonary edema, inflammation, or blood is present in a sample. Some of our results conflicted depending on the basis of reference. For example, in neonatal samples Nos. 8 and 9, as well as lung donor samples Nos. 15 and 16, mechanical ventilation may have induced inflammation and pulmonary edema. The same neonatal samples had congestion of the liver at autopsy. This may have decreased the activity when standardized against protein. Therefore, one has to be cautious in assessing the developmental profile of AOEs at the level of enzyme activity.
Whether coordinated regulation of various AOEs occurs in human tissues during development or in clinical conditions has not been defined. In addition, developmental changes of various AOEs are also complicated. In most animals, the expression of multiple AOEs increases in parallel toward term (27, 31). On the basis of our study we conclude that (1) pulmonary CAT activity and mRNA expression of MnSOD, CuZnSOD, and CAT increase toward term and adulthood; (2) hepatic activities of MnSOD and CuZnSOD and mRNA expression of MnSOD, CuZnSOD, and GPx increase toward adulthood; (3) the changes of AOE activities do not necessarily correlate with the corresponding mRNA levels; and (4) although significant correlations at the mRNA and activity levels were observed, our data do not support tightly coordinated regulation of AOEs in human lung or liver.
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Footnotes |
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Address correspondence to: Tiina M. Asikainen, M.D., Hospital for Children and Adolescents, Research Laboratory, University of Helsinki, Stenbäckinkatu 11, 00290 Helsinki, Finland. E-mail: tmasikai{at}cc.helsinki.fi
(Received in original form November 24, 1997 and in revised form April 13, 1998).
Abbreviations: antioxidant enzyme(s), AOE(s); catalase, CAT; complementary DNA, cDNA; copper-zinc superoxide dismutase, CuZnSOD; glyceraldehyde-3-phosphate dehydrogenase, GAPDH; glutathione peroxidase, GPx; manganese superoxide dismutase, MnSOD; messenger RNA, mRNA; superoxide dismutase, SOD.Acknowledgments: This study was supported by The Foundation for Pediatric Research in Finland, The Finnish Anti-Tuberculosis Association Foundation, The Sigrid Juselius Foundation, and The University of Helsinki. The cDNAs for all AOEs were kindly provided by Dr. Y.-S. Ho, Wayne State University, Detroit, MI.
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References |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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