1 Pediatric Nephrology, Yale University School of Medicine, New Haven 06520, and Albert Einstein College of Medicine, Bronx, New York 10467; and 2 Department of Medicine, Yale University School of Medicine, New Haven, Connecticut 06520; and Johns Hopkins School of Medicine, Baltimore, Maryland 21205
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ABSTRACT |
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Glucocorticoids (GC) regulate
Na-K-ATPase-subunit mRNA transcription. However, GC-induced increases
in Na-K-ATPase activity are not always paralleled by changes in subunit
mRNA abundance. We therefore examined posttranscriptional mechanisms of
subunit gene regulation by GC. cDNA-derived mRNAs encoding 1-,
3-, and
1-subunits were tested for stability and translation
efficiency in a cell-free lysate, in the presence of hydrocortisone
(HC) or dexamethasone (Dex). No effect of HC on subunit mRNA stability was noted. Translation efficiency of
1- and
3-mRNAs, but not of
1-mRNA, was significantly increased by HC and Dex. Deletion of the
5'untranslated region (5'UT) of
1-mRNA abolished this effect.
Translation of a chimeric
1-mRNA, constructed by transposing the
5'UT of
1 onto the coding region of
1, was enhanced by HC. Transposition of a putative steroid-modulatory element conserved in the
5'UT of all
isoforms (ACAGGACCC) onto the coding region of
1-mRNA rendered it responsive to HC. A synthetic primer containing the ACAGGACCC sequence abolished the effect of HC on
1- and chimeric
1-mRNAs. Our results indicate that GC can directly enhance
Na-K-ATPase translation in vitro in a subunit-specific manner, via a
putative GC-modulatory element situated in a predicted loop structure
within the 5'UT of
-mRNAs.
mRNA stability; 5'untranslated region; hormonal regulation of sodium-potassium-adenosinetriphosphatase; glucocorticoid modulatory element; mRNA secondary structure.
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INTRODUCTION |
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THE NA-K-ATPASE IS AN
INTEGRAL membrane protein present in all eukaryotic cells. It is
responsible for the ATP-dependent transport of sodium and potassium
across the cell membrane. The ionic gradients thus created are
essential for cell volume regulation, movement of ions and nutrients,
and electrical activity of excitable tissues (26, 31). It
is a heterodimer of two subunits in equal molar amounts. The
-subunit (Mr = 112,000) is catalytically
active, whereas the
-subunit (Mr = 35,000) facilitates the functional maturation and membrane insertion of
the
-heterodimer (26). Molecular cloning of the
cDNAs encoding the subunits has revealed that at least three isoforms
of the
-subunit (termed
1,
2, and
3) and two of the
-subunit (
1 and
2) exist, which exhibit complex
tissue-specific and developmental patterns of expression (19, 26,
31, 38, 44).
Regulation of Na-K-ATPase is especially critical in the kidney, where
the 1
1 unit is a major determinant of transepithelial sodium
transport across the tubular epithelium in all nephron segments
(15, 25). Mechanisms have evolved for short-term adaptation of the activity of existing pumps, and for long-term changes
in pump biosynthesis (3, 10, 15, 26, 31). Acute changes in
renal Na-K-ATPase biosynthesis can occur at the transcriptional (32, 39) and translational (13, 21) levels,
and may be driven by monovalent ions (5) or hormones such
as corticosteroids (4, 7-9, 17, 27, 42) and thyroid
hormone (23).
Glucocorticoids (GC) increase renal Na-K-ATPase activity
(16). This increase occurs in isolated perfused tubules
and in cultured renal epithelial cells, suggesting that it is
independent of changes in sodium load and hemodynamics. GC also
increase Na-K-ATPase-subunit protein abundance (2, 27),
and transcriptional regulatory mechanisms underlying these biosynthetic
changes are being actively investigated (7-9).
However, GC-induced increases in sodium pump activity are not always
paralleled by changes in subunit mRNA abundance. For instance, during
normal postnatal maturation, the upsurge in serum GC concentration is
associated with a doubling of Na-K-ATPase activity in the renal cortex,
but only a small increase in 1-mRNA levels (7). In
addition, the GC-induced increase in renal Na-K-ATPase activity in
adrenalectomized rats is prevented by inhibition of protein synthesis,
implicating translational control (2, 14). Furthermore, in
alveolar epithelial cells, dexamethasone (Dex) upregulates Na-K-ATPase
activity and
1-subunit protein abundance with no changes in the
1-mRNA abundance (2). Such observations suggest that
glucocorticoids may additionally regulate Na-K-ATPase-subunit
expression via posttranscriptional and/or translational mechanisms.
However, these downstream effects have hitherto been difficult to
assess in heterogeneous animal and cell culture systems that are
typically under the influence of multiple interdependent regulatory mechanisms.
In this study, we examined the effect of GC on Na-K-ATPase-subunit mRNA
stability and translation efficiency in a cell-free system. Our
findings indicate that GC can directly enhance Na-K-ATPase translation
in vitro in a subunit-specific manner, via a putative GC-modulatory
element situated in a predicted loop structure within the
5'untranslated region (5'UT) of -mRNAs.
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MATERIALS AND METHODS |
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All restriction enzymes were from New England Biolabs. Kits for in vitro transcription and translation were from Promega. L-[35S]methionine and [32P]dCTP were from Amersham. Enlightening was from New England Nuclear. PCR kit was from Perkin-Elmer Cetus. Cap analog and random-primed DNA labeling kit were from Boehringer Mannheim. Hydrocortisone (HC), Dex, and aldosterone were from Sigma.
Na-K-ATPase clones.
The Na-K-ATPase cDNA clones encoding full-length rat 1-,
3-, and
1-mRNAs have been previously described (13). The
construction of a variant
1-cDNA (termed
1-5'UTdel) devoid
of its 5'UT, and of a chimeric
1-cDNA (termed
1-chimera) with the
5'UT of
1 transposed onto the coding region of
1 have also been
published (13). A new variant
1-clone used in this
study (termed
1/GME) was constructed as follows. Comparison of
nucleotide sequences revealed a 9-bp segment (ACAGGACCC) in the 5'UT of
Na-K-ATPase
1-subunit (at nucleotide position
27 to
35), that is
conserved among all
-subunit isoforms across several species (see
Fig. 4A). This sequence is highly homologous to a
steroid-modulatory element identified in the 5'UT of myelin basic
protein mRNA (position
29 to
37), the translation efficiency of
which has previously been shown to be GC responsive (41).
A sense primer containing this segment, the Kozak consensus sequence,
and the first five residues of
1-mRNA were utilized to amplify the
entire coding region of
1 by standard PCR technique
(13). The PCR product was ligated into pGEM vector
(Promega), sequenced to confirm its identity, and termed
1/GME.
In vitro transcription. All cDNA clones were linearized with Sma I, and capped mRNAs were transcribed in vitro as described by the manufacturer (Promega). Transcription products were visualized by electrophoresis in 1%-formaldehyde agarose gels and quantitated by spectrophotometry. Comparable amounts of mRNA were obtained from each cDNA template. All in vitro transcribed mRNAs used in this study contain the entire coding regions and part of the untranslated regions, but are devoid of Poly(A) tails.
mRNA stability in reticulocyte lysate.
Equal amounts (200 ng) of capped mRNA transcripts derived from
full-length 1- and
1-cDNAs were incubated in 100 µl of rabbit reticulocyte lysate supplemented with [35S]methionine at
30°C for 60 min, with or without HC (10
8 M, a
physiologically relevant concentration). The mRNAs were extracted with
phenolchloroform, ethanol precipitated, and analyzed by formaldehyde
agarose gel electrophoresis, Northern blotting, and probing with random
primer-labeled full-length cDNAs for
1- or
1-subunit as
previously described (13).
In vitro translation. Equal amounts (200 ng) of each capped mRNA species were heated to 67°C for 10 min and translated at 30°C for 60 min in a 25-µl reaction mixture containing 15 µl of nuclease-treated rabbit reticulocyte lysate, 40 units of RNasin, 20 µM amino acids minus methionine, [35S]methionine (20 µCi per reaction), and varying concentrations of HC, Dex, or aldosterone as described in previous studies (2, 41). For some experiments, the reaction mixture was supplemented with canine pancreatic microsomes. Translation products were analyzed by SDS-PAGE followed by fixation and fluorography of the dried gel with Enlightening. We have previously shown that all Na-K-ATPase mRNAs are most efficiently translated at a concentration of 200 ng per reaction, and extending the reaction time or altering magnesium concentrations was devoid of any effect (13). Also, we have demonstrated that the addition of microsomes to the lysate did not alter the translation efficiency of Na-K-ATPase subunits (13). For the oligonucleotide competition experiments, a synthetic primer, ACAGGACCC (termed GME oligo, representing the putative GME), was added to the translation mixture (10-fold molar excess of primer to mRNA) at the start of the reaction. To assess the role of specific bases within the GME oligo, substitutions were incorporated based on conserved sequences (Fig. 4, A and B). The synthetic primer GCGGCACCC (termed mutant oligo) was tested in the translation mixture at a 10-fold molar excess of primer to mRNA.
Analysis of mRNA secondary structure.
The 5'UT sequence of 1-mRNA was subjected to secondary structure
analysis by using the FOLD program (13, 45), which
predicts the most stable secondary structure for the mRNA under study, by using thermodynamics and auxiliary information.
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RESULTS |
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Na-K-ATPase subunit mRNAs are stable in HC.
The capped mRNAs transcribed from full-length cDNAs encoding 1- and
1-Na-K-ATPase subunits were tested for stability in a
translationally active reticulocyte lysate mixture, in the presence of
physiological concentrations of HC. The
1-Na-K-ATPase cDNA yielded a
3.7-kb mRNA, and the
1-cDNA a predominant 2.3-kb mRNA species, along
with less abundant 1.7- and 1.4-kb messages (Fig. 1). This is consistent with the size of
native Na-K-ATPase subunit mRNAs in vivo, namely, 3.7 kb for
1 and
multiple species of 2.7, 2.3, 1.7, and 1.4 (ending at different
polyadenylation sites) for
1-mRNA (31). It should be
noted that the largest mRNA transcribed by our
1-cDNA is ~400 bp
smaller than the largest
1-mRNA transcribed in vivo, because part of
the 3'untranslated region is missing. However, we and others have
previously shown that, in spite of the varying sizes of the
1-mRNAs
transcribed, they are very efficiently and rapidly translated in
reticulocyte lysate to a single polypeptide of the predicted size and
immunoreactivity (13). We have also previously shown that
these mRNAs are stable in the lysate in the absence of HC
(13). No significant effect of HC on the stability of
either mRNA species was detected (Fig. 1). Therefore, the reticulocyte lysate represented an ideal system to explore the effects of GC on
Na-K-ATPase subunit mRNA translatability alone, independent of
transcriptional and mRNA stability considerations.
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Glucocorticoids enhance translation efficiency of - mRNAs.
We have previously demonstrated that cDNA-derived mRNAs encoding
1-,
3-, and
1-Na-K-ATPase subunits are reliably translated in
reticulocyte lysate into polypeptides of the predicted size and
immunoreactivity (13). In this study, we tested the
translation efficiency of
1-,
3-, and
1-Na-K-ATPase subunits
in the presence of various concentrations of HC, Dex, or aldosterone.
The concentrations of hormones tested were chosen on the basis of known
physiological plasma levels (7, 41) as well as previous in
vitro studies (2, 41). At the physiological concentrations
examined, both HC (Fig. 2A)
and Dex (not shown) selectively enhanced translation efficiency of
1- and
3-mRNAs but not of
1-mRNA. Densitometric analysis of
three separate experiments revealed an ~150% increase in
1- and
3-translation products in the presence of 10
6 and
10
10 M HC, and a 300% increase in 10
8 M HC
(Fig. 3). The addition of microsomes did
not alter these results for
1- or
1-mRNA quantitatively, although
the expected acquisition of core glycosylated sugars was apparent for
the
1-subunit (Fig. 2B).. In contrast,
addition of aldosterone at physiological or supraphysiological doses
was devoid of any effect on
1-mRNA translation (Fig. 2B), suggesting that the effect was specific to GC, and was unlikely to be
GC-receptor mediated.
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The 5'UT of 1-mRNA contains a transferable GC-modulatory
element.
To explore the potential role of 5'UT sequences, we examined the effect
of HC on translation efficiency of variant clones. In previous studies,
we have shown that translation of
1-mRNA devoid of the 5'UT
(
1-5'UTdel), proceeds efficiently in reticulocyte lysate
(13). The translation enhancing effect of HC on
1-mRNA was abolished by removal of the 5'UT (Fig.
4), suggesting the existence of a
GC-modulatory element within this sequence. Indeed, transposition of
the 5'UT of
1-mRNA onto the coding sequences of the previously
unresponsive
1-mRNA rendered the translation efficiency of the
chimeric
1-construct sensitive to physiological concentrations of HC
(Fig. 4). By densitometry, the abundance of chimeric
1-translation
product was increased by ~200% in 10
6 and
10
10 M HC, and ~350% in the presence of
10
8 M HC (Fig. 3). These results indicate that the
GC-modulatory element within the 5'UT of
1-mRNA can be transferred
to a normally unresponsive message.
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The sequence ACAGGACCC is a GC-modulatory element in 1-mRNA.
Comparison of nucleotide sequences revealed a 9-bp segment (ACAGGACCC)
at position
27 to
35 for the
1-isoform that is conserved in the
5'UT of all Na-K-ATPase
-subunit isoforms in several species (Fig.
5A). This sequence is absent
from the normally unresponsive
-subunits, but is highly homologous
to a steroid-modulatory element identified in the 5'UT of myelin basic
protein mRNA, the translation efficiency of which has previously been
shown to be GC sensitive (41). We therefore tested the
possibility that this segment may represent a GC-modulatory element,
initially by transposing it onto the coding region of
1-mRNA.
Indeed, the variant clone
1/GME produced a translation product of
the appropriate size, the abundance of which was increased by ~250%
in the presence of 10
8 M HC (Figs. 5B and 3).
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The 1-GC-modulatory element is situated in a loop structure.
Secondary structure examination of the 5'UT of
1-mRNA by the FOLD
program revealed that five of the nine residues representing the
GC-modulatory element are situated in a loop structure (Fig. 6), within the first of four such loops
previously described (13). Such loops are predicted to be
accessible to interacting proteins.
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DISCUSSION |
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In this study, we tested the hypothesis that GC influence
Na-K-ATPase-subunit mRNA expression via posttranscriptional mechanisms. The results demonstrate for the first time that physiological concentrations of GC enhance the translation efficiency of catalytic subunit mRNAs in vitro. This effect is specific to the -subunits, because
1-mRNA translation was unaltered by GC. In addition, this
property is restricted to the glucocorticoids, because aldosterone did
not influence
1-mRNA translation even at supraphysiological doses.
By using deletion mutants and chimeric clones, we established that the
5'UT of
1-mRNA contains a modulatory element that confers GC
responsiveness to the downstream coding sequences. By comparison of
nucleotide sequences, we identified a 9-bp segment, ACAGGACCC, that is
conserved across species within the 5'UT of all
-Na-K-ATPase mRNAs
but is absent from the
-subunit. This sequence is highly homologous
to the steroid modulatory element previously identified in myelin basic
protein mRNA, the translation of which is similarly steroid sensitive.
By creation of chimeric mutants as well as oligonucleotide competition
experiments, we have established that the sequence ACAGGACCC represents
a transferable GC-modulatory element within the 5'UT of
1-Na-K-ATPase mRNA, which is required to mediate the enhanced
translational response to GC. Analysis of predicted mRNA secondary
structure revealed that the GC-modulatory element is ideally situated
in a loop structure that is predicted to be accessible to interacting proteins.
Glucocorticoids mediate profound physiological and developmental effects in eukaryotes, by regulating the expression of several target genes (6). For the majority of proteins, this is achieved via a well-studied pathway involving specific intracellular glucocorticoid receptors and a cognate glucocorticoid response element in the promoter region of target genes. Several lines of evidence have endorsed the importance of this transcriptional mechanism in the regulation of Na-K-ATPase-subunit gene expression by GC (4, 7-9, 17, 27, 42, 43). However, additional posttranscriptional mechanisms must exist, because GC-induced increases in sodium pump activity are not always paralleled by changes in subunit mRNA abundance. These mechanisms have hitherto been difficult to ascertain in animal and cell culture systems where transcriptional regulation may predominate. In this study, a cell-free translation system was used to determine the direct effects of GC on translation efficiency of Na-K-ATPase subunit mRNAs synthesized in vitro, thereby circumventing any effects on transcription. In addition, the absence of sequences homologous to the cognate-glucocorticoid response element in the Na-K-ATPase mRNAs tested attests to the lack of transcriptional influences. Furthermore, the inability of aldosterone to mimic the effect of GC, even at supraphysiological doses, suggests that the enhanced Na-K-ATPase translation noted in this study is not receptor mediated.
The posttranscriptional regulation of gene expression by GC may occur
either through mRNA stabilization or via direct effects on translation.
For example, Dex enhances Bcl-x gene expression by significantly
extending mRNA stability (11). Although we have not
performed a detailed kinetic assay of Na-K-ATPase mRNA stability, our
results clearly indicate that both subunit mRNAs were equally stable in
the lysate during the translationally active period, irrespective of
the presence or absence of GC. Thus we were able to exclude any effects
of GC on mRNA stability as a potential explanation for the enhanced
translation of Na-K-ATPase -mRNAs that we have observed. However,
these in vitro studies do not rule out the potential for additional
effects of GC on mRNA stability in nucleated cells. Indeed, the mRNAs
used in this in vitro study were devoid of Poly(A) tails. In vivo, it
is possible that the stability of native mRNAs, with their complete
Poly(A) tails and all polyadenylation signals, may be significantly
influenced by physiological concentrations of GC. This may be
especially relevant to Na-K-ATPase-
1 mRNA translation in vivo,
because the largest mRNA transcribed in vitro lacked ~400 bp of
3'untranslated sequences.
Several recent reports have indicated that GC can directly influence
mRNA translation, either positively or negatively. For instance, Dex
inhibits translation of inducible nitric oxide synthase (29), cyclooxygenase-2 (34), cytokines such
as leukemia-inhibitory factor (22), and tumor necrosis
factor- (18). On the other hand, Dex has been shown to
play a role in the enhanced translation efficiency of a variety of
polypeptides, including myelin basic protein (41), myelin
proteolipid protein (41), vasopressin receptor
(12), glutamine synthetase (37),
2-adrenergic receptor (33) and, most recently, the
proximal tubule Na/H antiporter (1). Perhaps the
best-studied example is that of myelin basic protein, the translation
efficiency of which was enhanced two- to threefold in a cell-free
system in the presence of physiological concentrations of HC or Dex
(41). This effect was mediated by a steroid- modulatory
element situated in a predicted loop structure at position
29 to
37
within the 5'UT of myelin basic protein mRNA. It is interesting to note
the several similarities between that study and ours. In the present
report, the GC-induced threefold increase in translation efficiency of
1-Na-K-ATPase mRNA was also mediated by a highly homologous
GC-modulatory element in a predicted loop at position
27 to
35
within its 5'UT. Furthermore, a comparison of putative glucocorticoid
modulatory elements within the 5'UTs of
-Na-K-ATPases across
isoforms and species reveals the striking and complete conservation of
the very same nucleotides that have been shown by site-directed
mutational analysis to be critical for GC responsiveness in myelin
basic protein (highlighted by asterisks in Fig. 4A). Indeed,
the present study has demonstrated, by oligonucleotide-competition
experiments, that the same nucleotides are also necessary for the
translational control of
-Na-K-ATPase by GC.
The concentrations of GC that were found to be effective in this study
are within the physiological range (41) and have been used
in previous studies. For instance, during postnatal maturation of the
kidney, a significant increase in Na-K-ATPase activity (paralleled by
only a small increase in catalytic-subunit mRNA level) is associated
with a measured plasma-glucocorticoid level in the 108 M
range (7). In addition, the Dex-induced upregulation of Na-K-ATPase protein abundance and activity (with no change in
1-mRNA
level) in alveolar epithelial cells has been demonstrated to occur at
GC concentrations of 10
7 and 10
8 M
(2). In our study, the maximal stimulatory effect on
1-mRNA translation was noted at GC concentrations of
10
8 M (3-fold increase); concentrations of
10
6 or 10
10 M yielded only modest increases
(1.5-fold). Our results suggest that, in addition to the
well-documented receptor-mediated transcriptional regulation of
Na-K-ATPase by GC, direct translational control may represent an
adjunctive, but physiologically relevant, mechanism for the basal,
constitutive expression of Na-K-ATPase.
Regulation of gene expression at the level of translation is now widely
appreciated as an important modulator of protein synthesis (24,
35). Elucidation of the ribosome-scanning model has shown that
the peptide chain initiation phase is the rate-limiting step in protein
translation (28, 36). Efficiency of translation initiation
is heavily influenced by mRNA secondary structure of the 5'UT
(28), and by the interactions between a variety of proteins that bind either to the 5'UT of mRNAs (20, 40) or directly to components of the translational machinery
(30). Pertinent to this study, the proximity of the
putative GC-modulatory element to the initiator methionine, combined
with the fact that most of the known physiological effects on
translation are exerted at the level of polypeptide chain initiation
(35), suggests that GC increase translation of
1-Na-K-ATPase by enhancing the formation of the initiation complex.
Can enhanced translation of Na-K-ATPase -mRNAs (but not
-mRNA)
lead to increased activity of the
heterodimer? We have previously shown that the translation efficiency of
-mRNAs is markedly greater than that of
-mRNAs, both in vitro and in vivo (13). It is therefore reasonable to speculate that newly
synthesized
-subunit protein is normally present in enough excess to
incorporate all the
-protein made, including the enhanced amounts of
-protein induced by glucocorticoid surges, thereby resulting in
increased pump activity. Furthermore, our study does not exclude any
additional effects of GC on Na-K-ATPase activity from enhanced
trafficking of the heterodimer, as has been suggested for the
Na+/H+ exchanger (1).
In summary, we have shown that GC can directly enhance Na-K-ATPase
translation in vitro in a subunit-specific manner, via a putative
GC-modulatory element in a loop structure within the 5'UT of -mRNAs.
Several speculations regarding the physiological significance of these
findings can be offered. First, translational control may represent an
adjunctive mechanism for the constitutive, basal regulation of
Na-K-ATPase function by GC. Second, such a mechanism could account for
instances where GC-induced increases in sodium pump activity are not
accompanied by changes in subunit mRNA abundance. Third, it may provide
for a mechanism by which GC can regulate Na-K-ATPase-gene expression
during the physiological GC surge characteristic of early postnatal
development of several organ systems. Fourth, it may represent a
general mechanism by which growth-related proteins (such as Na-K-ATPase
and myelin basic protein) could rapidly overcome a state of repressed
translation. We have previously shown that translation efficiency of
the Na-K-ATPase
-subunit is chronically inhibited by its complex
5'UT secondary structure (13), reminiscent of the
constitutionally repressed translation of growth-related proteins. It
is therefore possible that the appearance of growth stimuli such as
steroids could counteract the repressive influences on the 5'UT,
perhaps by altering the phosphorylation state of translation regulatory
molecules, thereby allowing for rapid translation of the proteins at a
crucial period during altered cellular growth and development. It will
be important in future experiments to document a direct mRNA-protein
interaction between steroids and the GC-modulatory element, and to
identify the critical residues within this element that mediate this
interaction. It will also be of value to test the behavior of variant
Na-K-ATPase clones in transfected cells, to search for similar
translational effects of GC in vivo.
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ACKNOWLEDGEMENTS |
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This work was supported by grants from the National Institutes of Health to P. Devarajan (DK-47072, DK-53289) and E. J. Benz, Jr. (HL-24385, HL-44985, HL-23076).
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FOOTNOTES |
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Address for reprint requests and other correspondence: P. Devarajan, Children's Hospital at Montefiore, Albert Einstein College of Medicine, 111 East 210th St., Bronx, NY 10467 (E-mail: pdevaraj{at}aecom.yu.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 3 February 2000; accepted in final form 11 August 2000.
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