Departments of 1 Molecular Genetics, Biochemistry, and Microbiology, 2 Molecular and Cellular Physiology, and 3 Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio, 45267; and 4 Department of Biological Sciences, Northern Kentucky University, Highland Heights, Kentucky 41099
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ABSTRACT |
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To study the role of
Na+/H+ exchanger isoform 2 (NHE2) and isoform 3 (NHE3) in sodium-fluid volume homeostasis and renal
Na+ conservation, mice with Nhe2
(Nhe2/
) and/or Nhe3
(Nhe3
/
) null mutations were fed a
Na+-restricted diet, and urinary Na+ excretion,
blood pressure, systemic acid-base and electrolyte status, and renal
function were analyzed. Na+-restricted
Nhe2
/
mice, on either a wild-type or
Nhe3 heterozygous mutant (Nhe3+/
)
background, did not exhibit excess urinary Na+ excretion.
After 15 days of Na+ restriction, blood pressure,
fractional excretion of Na+, and the glomerular filtration
rate (GFR) of
Nhe2
/
Nhe3+/
mice
were similar to those of Nhe2+/+ and
Nhe3+/
mice, and no metabolic disturbances
were observed. Nhe3
/
mice maintained on a
Na+-restricted diet for 3 days exhibited hyperkalemia,
urinary salt wasting, acidosis, sharply reduced blood pressure and GFR,
and evidence of hypovolemic shock. These results negate the hypothesis that NHE2 plays an important renal function in sodium-fluid volume homeostasis; however, they demonstrate that NHE3 is critical for systemic electrolyte, acid-base, and fluid volume homeostasis during
dietary Na+ restriction and that its absence leads to renal
salt wasting.
sodium absorption; sodium/hydrogen exchanger; slc9a2; slc9a3
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INTRODUCTION |
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MAINTENANCE OF
NA+-fluid volume homeostasis by the kidney requires
the tightly regulated activities of a number of Na+
transport proteins. One of the most important, in terms of bulk reabsorption of Na+ with accompanying fluid, is
Na+/H+ exchanger isoform 3 (NHE3) (14,
27), which is expressed in brush-border membranes of the
proximal tubule and at lower levels in the thick ascending limb
(1, 3). By transporting Na+ into the cell in
exchange for H+, NHE3 reabsorbs large quantities of both
Na+ and HCO
Null mutant homozygous (Nhe3/
) mice have low
blood pressure, high-renin mRNA in kidney, and sharply elevated serum
aldosterone levels, consistent with a chronic volume-depleted state
(19). Nhe3
/
mice exhibit severe
absorptive defects in both the kidney and intestine (9, 19,
29). In situ microperfusion studies of the proximal tubule
demonstrated that reabsorption of both fluid, which accompanies
Na+, and HCO
) mice but that
fluid delivery to the distal convoluted tubule was the same as that of
wild-type mice (9). In the knockout, this was due largely
to a reduction in the glomerular filtration rate (GFR), and in
Nhe3+/
mice it was due to increased absorption
in the loop segment. Nhe3
/
mice are mildly
acidotic, which may be due to both the HCO
The studies discussed above have shown that the loss of NHE3 impairs
Na+ handling in the renal proximal tubule and
Na+-fluid volume homeostasis. However, at least part of the
fluid volume deficit in Nhe3/
mice is likely
to be due to the chronic diarrheal state; the ability of the
NHE3-deficient kidney to retain Na+ in vivo has not been
rigorously assessed and, therefore, the relative contribution of the
renal defect to the Na+-fluid volume deficit is unclear.
Also unknown is whether NHE2 plays a significant role in renal
Na+ conservation under normal conditions or provides
compensation for the loss of NHE3. The phenotype of Nhe2
null mutant (Nhe2
/
) mice has yielded no
evidence of a deficit in renal function (18);
nevertheless, it is possible that NHE2 plays a supplementary role in
Na+ reabsorption. To address these issues, and to further
assess any metabolic perturbations caused by the loss of these
exchangers, we subjected Nhe2
/
and
Nhe3
/
mice to dietary Na+
restriction and analyzed urinary Na+ excretion and renal function.
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METHODS |
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Production of mutant mice and analysis of genotypes.
Homozygous mutant mice with null mutations in the Nhe2 or
Nhe3 genes and the corresponding wild-type control mice were
obtained by breeding of heterozygous Nhe2 or Nhe3
mutant mice developed in previous studies (18, 19). For
the study described in Table 1 and Fig.
2, doubly heterozygous mutant
(Nhe2+/Nhe3+/
)
mice were bred to obtain mice that were null mutant or wild-type with
respect to the Nhe2 gene but on an Nhe3
heterozygous mutant (Nhe3+/
) background.
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Dietary sodium restriction and analysis of urine and feces.
Adult mice were housed in metabolic cages and provided with drinking
water and food ad libidum, as described previously (20). The food (Harlan Tecklad, Madison, WI) contained normal (1% NaCl), medium (0.1% NaCl), or low (0.01% NaCl) amounts of
Na+. In the studies with
Nhe2/
mice, 1-day urine and fecal samples
were collected. In the studies with
Nhe2
/
Nhe3+/
mice,
urine and fecal samples were each pooled over a 3-day period. In the
studies with Nhe3
/
mice, 1-day urine samples
were collected; feces were not collected because of diarrhea, and cages
were cleaned and urine was collected several times a day to avoid
contamination of urine with feces. In each experiment, the volume of
urine was measured, urinary Na+ and K+
concentrations were analyzed by flame photometry (Corning model 480),
and total excretion of each ion was calculated. Feces were collected
and homogenized in 4-10 ml of 0.75 N nitric acid. After overnight
incubation, the tubes were centrifuged and the supernatants were
analyzed to determine fecal Na+ and K+ excretion.
Analysis of urinary Ca2+ excretion. Urine volumes of adult mice maintained on a diet containing 1% NaCl were measured, Ca2+ concentrations were determined colorimetrically (arsenazo III assay, Sigma), and total urinary Ca2+ excretion was calculated.
Cardiovascular and renal measurements.
After either 15 (for
Nhe2/
Nhe3+/
mice)
or 3 days (for Nhe3
/
mice) of sodium
depletion, the mice were anesthetized with intraperitoneal injections
of inactin (100 µg/g body wt) and ketamine (50 µg/g body wt) and
surgically instrumented for cardiovascular and renal measurements under
baseline conditions and after extracellular volume expansion (ECVE), as
described previously (4, 9, 12). Mean arterial blood
pressure and heart rate were monitored via a pressure transducer
connected to a catheter in the femoral artery, and urine was collected
via a catheter in the bladder (9). Urine electrolytes were
measured by flame photometry, and plasma electrolytes and acid-base
status were analyzed using a pH/blood-gas analyzer (model 348, Chiron
Diagnostics; Norwood, MA). Two 30-min baseline clearance measurements
were performed in the presence of a maintenance infusion of isotonic
saline (0.15 µl/g of body wt/min) and were followed by two additional
30-min clearance periods in which isotonic saline was infused more
rapidly (1.0 µl/g of body wt/min) to induce ECVE. GFR under baseline
and ECVE conditions was determined using fluorescein
isothiocyanate-labeled inulin (1.5 µg/g of body wt/min;
9) and was averaged for each of the two clearance periods.
For each mouse, the values for mean arterial pressure and heart rate
were an average of the values recorded during the last 2 min of the two
collection periods that occurred either before or after ECVE. Blood for
analysis of plasma electrolytes and acid-base status was taken at the
midpoint of each 30-min collection period; values for individual mice
were an average of the two samples taken either before or after ECVE.
Statistics. Data are presented as means ± SE. Student's t-test was used to compare each group of mutant mice to the corresponding control mice.
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RESULTS |
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Loss of NHE2 does not lead to renal salt wasting during dietary
Na+ restriction.
As an initial test of the hypothesis that NHE2 contributes to renal
Na+ conservation, wild-type and
Nhe2/
mice were maintained successively on
diets containing high, medium, and low concentrations of sodium (1%
NaCl for 6 days, followed by 0.1% NaCl for 5 days, and then 0.01%
NaCl for 5 days), and urinary excretion of both Na+ (Fig.
1A) and K+ (Fig.
1B) was analyzed. On each of the three diets, the amount of
Na+ and K+ excreted in the urine was similar
for Nhe2+/+ and Nhe2
/
mice. Even when fed a 0.01% NaCl diet,
Nhe2
/
mice were able to reduce urinary
Na+ excretion to very low levels
(Nhe2+/+, 7.3 ± 1.6 µmol/day;
Nhe2
/
, 6.7 ± 1.7 µmol/day),
indicating that the NHE2-deficient kidney is able to retain
Na+ as well as that of wild-type mice.
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Acid-base and electrolyte status, mean arterial pressure, and renal
function in Na+-restricted NHE2-deficient
mice.
After the 15-day period of dietary Na+ restriction, the
Nhe2+/+Nhe3+/ and
Nhe2
/
Nhe3+/
mice
were anesthetized and their blood pressure, acid-base and electrolyte
status, and renal function were assessed under baseline conditions and
after ECVE. As shown in Table 1, baseline values for mean arterial
blood pressure, heart rate, plasma Na+, plasma
K+, and blood pH and HCO
and
Nhe2
/
Nhe3+/
mice.
The GFR was similar in both groups under baseline conditions and did
not change significantly after volume expansion (Table 1). Renal
K+ handling was similar in the two groups under baseline
conditions, and K+ excretion increased in a similar fashion
in both groups after volume expansion. There was no major difference in
renal Na+ handling between the two groups under either
experimental condition (Table 1). Under baseline conditions, the mean
value for fractional Na+ excretion was slightly higher in
Nhe2
/
Nhe3+/
mice
than in Nhe2+/+Nhe3+/
mice; however, the difference was not statistically significant, and
both values were very low, with greater than 99.9% of the filtered
Na+ being reabsorbed. After ECVE, fractional
Na+ excretion increased sharply to similar values in both
groups of mice. These data provide no evidence of an important role for NHE2 in either renal Na+ reabsorption or the response to ECVE.
Nhe3/
mice exhibit mild renal salt wasting and do
not tolerate a Na+-restricted diet.
Micropuncture (10) and in situ microperfusion
(30) studies of Nhe3
/
mice have
shown that the loss of NHE3 causes a severe impairment of
Na+ reabsorption in the proximal tubule; however, it is
unclear whether this leads to significant renal salt wasting. To
examine this issue, we maintained Nhe3+/+ and
Nhe3
/
mice on a Na+-replete diet
for 4 days and then switched them to a Na+-restricted
(0.01% NaCl) diet for 3 days. Their body weights and urinary
Na+ and K+ excretion were measured daily. Fecal
excretion of Na+ and K+ were not determined
because the diarrheal state of Nhe3
/
mice
makes it difficult to obtain accurate measurements, and these data were
not necessary for assessment of renal salt wasting.
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Acid-base and electrolyte status, mean arterial pressure, and renal
function in Na+-restricted NHE3-deficient
mice.
The sharp drop in body weight by day 3 of dietary
Na+ restriction indicated that
Na+-restricted Nhe3/
mice were
unable to maintain Na+-fluid volume homeostasis. The mice
were anesthetized and surgically prepared for analysis of mean arterial
pressure, systemic acid-base and electrolyte status, and renal function
(Figs.
5-7
and Table 2). Three of the
eight Nhe3
/
mice that survived during the
3-day period of dietary Na+ restriction died shortly after
being anesthetized for surgery, presumably as a result of hypovolemic
shock.
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Urinary calcium excretion is similar in Nhe2/
,
Nhe3
/
, and wild-type mice.
Loss of the apical Na+-K+-2Cl
cotransporter of the thick ascending limb or the thiazide-sensitive
NaCl cotransporter of the distal convoluted tubule causes
hypercalciuria or hypocalciuria, respectively (20-22,
26). To explore the possibility that null mutations in NHE2 or
NHE3 might affect renal calcium handling, we maintained null mutants
and wild-type controls on a 1% NaCl diet and analyzed urinary calcium
excretion. As shown in Fig. 8, there were
no significant differences between the knockouts and their wild-type
controls.
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DISCUSSION |
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Previous studies of Nhe2/
and
Nhe3
/
mice showed that NHE3 is the major
absorptive Na+/H+ exchanger in the kidney
(9, 19, 29) but did not reveal a renal function for NHE2
(18). It was unclear, however, whether the loss of NHE3
caused significant renal salt wasting and whether NHE2 might play a
supplementary role in Na+ reabsorption. To gain a better
understanding of the relative roles of NHE2 and NHE3 in renal
Na+ conservation, we used the NHE2 and NHE3 knockouts to
determine whether the loss of either exchanger would result in urinary
salt wasting or metabolic disturbances when the mice were fed a
Na+-restricted diet.
NHE2-deficient mice exhibit a stomach phenotype characterized by
achlorhydria and the loss of gastric parietal and chief cells (18). Although NHE2 mRNA is expressed in mouse kidney at
levels comparable to those observed in stomach and is expressed at high levels in the small intestine and colon, renal and intestinal phenotypes were not observed in Nhe2/
mice.
Because NHE2 has been localized to apical membranes of the thick
ascending limb and distal convoluted tubule (5, 25), a
renal absorptive function seemed likely. NHE2 has also been identified
in basolateral membranes of the inner medullary collecting duct cell
line (IMCD3) (23), consistent with functions such as
secretion or cell volume and pH regulation.
Nhe2
/
mice maintained on a
low-Na+ diet were able to retain Na+ as well as
wild-type mice; however, similar studies of mice lacking the
thiazide-sensitive Na+-Cl
cotransporter (NCC)
also failed to reveal a salt-wasting phenotype (20),
despite the known function of NCC in NaCl absorption in the distal
convoluted tubule. Therefore, these results did not exclude the
possibility that the loss of NHE2 causes a mild impairment of
Na+ reabsorption but that other Na+
transporters provide compensation for the defect.
It was shown previously that Na+ reabsorption is reduced in
the proximal tubule of Nhe3+/ mice and that
compensation occurs via increased absorption in the loop segment rather
than a decrease in GFR, as in Nhe3
/
mice
(9). This suggested that under Na+-depleted
conditions a reduction in NHE3-mediated Na+ reabsorption in
the proximal tubule might stress the more distal Na+
reabsorption mechanisms and reveal a contribution by NHE2. However, Nhe2
/
Nhe3+/
mice
subjected to dietary Na+ restriction retained
Na+ as well as
Nhe2+/+Nhe3+/
mice and
exhibited none of the metabolic defects observed in Nhe3
/
mice. Measurements of renal function
revealed no alterations in GFR, fractional excretion of
Na+, or the natriuretic response to ECVE. Also, in contrast
to mice lacking NCC (20), when fed a
Na+-restricted diet they did not exhibit a decrease in
blood pressure indicative of a defect in Na+-fluid volume
homeostasis. On the basis of these results, which are in sharp contrast
to those obtained with the NHE3 knockout, we conclude that NHE2 plays
little if any role in Na+-fluid volume homeostasis.
Similarly, there was no indication of a role for NHE2 in renal
HCO/
and
Nhe2
/
Nhe3+/
mice.
Previous studies of Nhe3
/
mice, in which
inhibitors of Na+/H+ exchange were utilized,
also yielded no evidence of a role for NHE2 in HCO
/
mice, which normally exhibit a mild acidosis (19, 29). If NHE2 played a major role in renal HCO
/
Nhe3
/
mice would have been expected to have a more severe acidosis. Although
additional studies will be needed to assess the possible function of
NHE2 in renal HCO
The previous demonstration of an absorptive defect in
Nhe3/
proximal tubules (19, 29)
suggested that at least part of the fluid volume deficit was due to
renal salt wasting, with an additional component resulting from the
intestinal defect. However, because a defect in bulk reabsorption of
Na+ in the proximal tubule can be sensed by the macula
densa, it remained possible that tubuloglomerular feedback (TGF) might
bring about a sufficient reduction in GFR to enable the
Na+-transport mechanisms of the more distal segments of the
nephron to reduce urinary Na+ losses to the low levels
observed in wild-type mice. Previous studies showed that TGF was intact
in Nhe3
/
mice, that GFR was reduced to about
65-70% of normal, and that fluid delivery to the distal tubule
was the same as in wild-type mice (4, 9). Surprisingly,
absorption in the loop segment and apical
Na+-K+-2Cl
cotransporter (NKCC2)
protein levels was reduced in the knockout (4, 9), rather
than upregulated as would be expected if NKCC2 provided partial
compensation. Thus it was unclear whether the loss of NHE3 caused
severe renal salt wasting, as observed in null mutants for NKCC2
(21, 26) and the epithelial Na+ channel (ENaC)
(2, 6, 8, 11, 15, 25).
To examine this issue, we subjected NHE3-deficient mice to dietary
Na+ restriction. During the control period, water intake
was higher in Nhe3/
mice than in wild-type
mice and, although it decreased during Na+ restriction, it
remained higher than wild-type levels. Part of the elevation in water
intake was undoubtedly secondary to the diarrheal state, which was
noticeably diminished during Na+ restriction. Mean urinary
volume was significantly higher in the knockout during Na+
restriction, and the greatest increase was observed in
Nhe3
/
mice that exhibited relatively strong
Na+ retention. Na+ restriction could not be
extended beyond 3 days because of severe weight loss, indicative of
volume depletion, and morbidity (2/10 mice died) that occurred in the
NHE3 knockout. After the switch to the Na+-restricted diet,
the rate of reduction in urinary Na+ excretion was much
less in the knockout than in wild-type mice. This may be a reflection
of the fact that the more distal Na+-conserving mechanisms
were already upregulated in the knockout mice when the switch in diet
was made, whereas wild-type mice have a substantial reserve capacity
that can be activated during the days after the switch. By the third
day of Na+ restriction, urinary losses of Na+
were four to five times greater in the knockout mice, demonstrating that the loss of NHE3 causes urinary salt wasting.
When we analyzed renal function after 3 days of Na+
restriction, three of the eight surviving knockout mice died during
anesthesia and surgical instrumentation. Given the magnitude of the
weight loss and reduced blood pressure, the observed deaths were
probably due to hypovolemic shock. Among the remaining mice, the
response was variable. Some of the knockouts had very low GFRs and were in hypovolemic renal failure; however, under baseline conditions two of
them had GFRs within the range observed for knockouts on a normal-
Na+ diet (4), but with blood pressure and
fractional Na+ excretion within the range observed for
wild-type mice after 3 days of Na+ restriction. It should
be noted that 3 days of Na+ restriction is a quite brief
period and not long enough for Na+-conserving mechanisms to
be fully activated in wild-type control mice (for example, see Table
1). Thus the degree of renal Na+ conservation in the
knockout, in which Na+-conserving mechanisms are sharply
elevated even when maintained on a normal diet (19), falls
far short of that of wild-type mice. NHE2-deficient or wild-type mice
exhibit no ill effects when maintained for several weeks on the
low-Na+ diet used in this study, whereas
Nhe3/
mice have little tolerance for even a
brief period of Na+ restriction. These observations
demonstrate that NHE3, in sharp contrast to NHE2, is critically
important for Na+-fluid volume homeostasis during salt deprivation.
Defective Na+-fluid volume homeostasis resulting from genetic defects in renal Na+ transporters leads to a number of metabolic disorders. NCC and NKCC2 mutations are associated with hypokalemic alkalosis (21, 22), and ENaC mutations are associated with hyperkalemic acidosis (6). Previous studies showed that Na+-replete NHE3-deficient mice are mildly acidotic and hyperkalemic (19, 29), and the present study shows that dietary Na+ restriction leads to reduced plasma Na+ and a worsening of both the metabolic acidosis and hyperkalemia. In contrast, no metabolic disturbances were observed in NHE2-deficient mice under either Na+-replete or Na+-restricted conditions, consistent with the lack of an effect on Na+-fluid volume homeostasis.
Hyperkalemia in ENaC-deficient mice has been attributed to reduced
K+ secretion via apical channels in the collecting duct,
which are normally coupled with Na+ reabsorption via ENaC
(11). Similarly, hypokalemia in humans with NKCC2 or NCC
mutations has been proposed to be due to a compensatory increase in
ENaC activity, with a coupled increase in K+ secretion
(21, 22). However, NHE3-deficient mice would be expected
to have increased ENaC activity, as indicated by increased aldosterone
levels (19) and increased abundance of the 70-kDa form of
the -subunit (4). Because this would be expected to lead to increased K+ secretion and subsequent hypokalemia,
the basis for the hyperkalemia in Nhe3
/
mice
is unclear.
Loss of the apical Na+-K+-2Cl
cotransporter of the thick ascending limb or the thiazide-sensitive
NaCl cotransporter of the distal convoluted tubule causes
hypercalciuria or hypocalciuria, respectively (20-22,
26). It has been suggested that the loss of Na+
uptake activity in the distal convoluted tubule as a result of NCC null
mutations increases the driving force for Ca2+ absorption,
thereby leading to reduced urinary Ca2+ excretion
(21). Because NHE2, like NCC, has been reported to be
expressed in apical membranes of distal convoluted tubule cells (5), it seemed possible that hypocalciuria would be
observed, but this was not the case. The observation that
NHE2-deficient mice have normal urinary Ca2+ excretion
provides suggestive evidence that NHE2 is not involved in
Na+ reabsorption in the distal convoluted tubule. With
regard to NHE3, we anticipated that its absence might lead to
hypercalciuria as a result of compensatory upregulation of
Na+ reabsorption via NCC in the distal convoluted tubule or
reduced paracellular transport of Ca2+ in the proximal
tubule due to the observed reduction in fluid transport
(29); however, urinary Ca2+ excretion in
Nhe3
/
mice was the same as that of wild-type mice.
Despite the fact that NHE3 is a major bulk transporter of
Na+ in the kidney, the salt-wasting defect seems mild when
compared with that of NKCC2 and ENaC null mutant mice (2, 8, 11, 15, 26). Although ENaC is responsible for only a small fraction of total Na+ reabsorption, its activity in the collecting
duct is critical because there are no downstream transporters that can
compensate for its absence and it is located beyond the macula densa,
where TGF can adjust GFR (16). The severe phenotype of the
NKCC2 knockout is due to the loss of NaCl reabsorption in the thick
ascending limb and the urinary concentrating defect, and it is
conceivable that an impairment of the TGF mechanism (10, 16,
17) also contributes to the defect. The mild perturbation of
Na+-fluid volume homeostasis in NCC mutants
(20) is presumably because this transporter does not
reabsorb large quantities of Na+ and because absorption via
ENaC can compensate for the defect. The results of the present and
previous studies of Nhe3/
mice (9, 19,
29) suggest that the loss of NHE3, which normally mediates bulk
reabsorbtion of Na+, is tolerated when mice are maintained
on a normal diet because reduced blood pressure and TGF limits the
amount of Na+ that is filtered and distal mechanisms of
Na+ reabsorption are intact. However, when they are fed a
Na+-restricted diet, the reduction in GFR and induction of
Na+-conserving mechanisms in more distal segments of the
nephron are not sufficient to fully compensate for the defect in bulk Na+ reabsorption in the proximal tubule.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-50594, DK-57552, and DK-54430.
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FOOTNOTES |
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Address for reprint requests and other correspondence: G. E. Shull, Dept. of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, 231 Albert Sabin Way, ML 524, Cincinnati, OH 45267-0524 (E-mail: shullge{at}ucmail.uc.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 20 March 2001; accepted in final form 23 March 2001.
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