Agonist-induced translocation of Gq/11
immunoreactivity directly from plasma membrane in MDCK
cells
John M.
Arthur1,
Georgiann P.
Collinsworth2,
Thomas W.
Gettys2, and
John R.
Raymond2
1 University of Louisville,
Louisville, Kentucky 40202; and
2 Medical University of South
Carolina, Charleston, South Carolina 29425
 |
ABSTRACT |
Both
Gs
and
Gq
are palmitoylated and both
can move from a crude membrane fraction to a soluble fraction in
response to stimulation with agonists. This response may be mediated
through depalmitoylation. Previous studies have not demonstrated that
endogenous guanine nucleotide-binding regulatory protein (G protein)
-subunits are released directly from the plasma membrane. We have
examined the effect of agonist stimulation on the location of
Gq/11
immunoreactivity in Madin-Darby canine kidney (MDCK) cells. Bradykinin (BK; 0.1 µM)
caused Gq/11
, but not
Gi
, to rapidly translocate from
purified plasma membranes to the supernatant. AlF and GTP also caused
translocation of Gq/11
immunoreactivity from purified plasma membranes. BK caused
translocation of Gq/11
immunoreactivity in
intact cells from the basal and lateral plasma membranes to an
intracellular compartment as assessed by confocal microscopy. Thus
Gq/11
is released directly from
the plasma membrane to an intracellular location in response to
activation by an agonist and direct activation of G proteins. G protein
translocation may be a mechanism for desensitization or for signaling specificity.
G protein; bradykinin; palmitoylation
 |
INTRODUCTION |
HETEROTRIMERIC guanine nucleotide-binding regulatory
proteins (G proteins) couple cell surface receptors to intracellular second messengers. The
-subunit is covalently bound to a myristoyl (2, 9) and/or a palmitoyl (12) side chain. These fatty acyl side chains
aid in membrane association and signaling (9, 34). Whereas some G
proteins are both myristoylated and palmitoylated, Gq/11
and
Gs
are only palmitoylated (35).
As early as 1985, Rodbell proposed that G protein
-subunits might
diffuse from the plasma membrane after activation to function as
"programmable messengers" (26). That
Gs
can translocate from a
membrane fraction to the soluble phase after activation has been well
documented, although no evidence of translocation of endogenously
expressed G proteins directly from the plasma membrane, where the G
protein would be likely to encounter an agonist-liganded receptor, has yet been provided. Two lines of evidence suggest that G proteins can
move from one compartment of the cell to another. First,
Gs
can translocate from crude
cell particulate fractions to cytoplasm after agonist stimulation.
Activation of Gs
by several
agents has been shown to increase the cytosolic content of
Gs
in S49 and mouse mastocytoma
cells (11, 21, 23) and decrease the Gs
immunoreactivity in crude
membrane fractions by up to 75% (16, 22). Although those studies show
an agonist-induced dissociation of
Gs
from a cellular particulate
fraction, a definite identification of the plasma membrane as the
source of the G proteins has not yet been established. Recently,
translocation of transfected HA-tagged Gs
from the plasma membrane in
response to
-adrenergic receptor stimulation has been shown by
immunofluorescence microscopy (33).
Similarly, members of the Gq/11
family have also been shown to translocate in intact cells. Levels of
Gq
immunoreactivity in crude
membrane fractions have been shown to decrease after 16-h treatment
with carbachol in CHO cells (18) and gonadotropin-releasing hormone
(GnRH) in pituitary cells (29). In HEK cells transfected with G11
subunits and
thyrotropin-releasing hormone (TRH) receptors, stimulation with TRH
caused a time-dependent increase in
G11
immunoreactivity in the
cytosolic fraction and low-density membranes and a decrease in
G11
immunoreactivity in plasma
membrane fractions (30). Total
G11
immunoreactivity decreased
during this time. In CHO cells transfected with muscarinic receptors,
stimulation with carbachol for 30 min resulted in a decrease in
Gq
and
G11
immunoreactivity in a
plasma membrane fraction and an increase in the light vesicular
membrane fraction (31). A second line of evidence supporting the
functional importance of membrane attachment of G proteins derives from
studies of their lipid anchors. Lipid anchors have been shown to be
important for targeting and maintenance of
Gs
and
Gq
in cellular particulate
fractions (13, 34). Stimulation of
2-adrenergic receptors in
COS and S49 cells induces a dynamic cycle of palmitoylation and
depalmitoylation of Gs
(5, 19,
32). The importance of membrane attachment is underscored by a study in
which cysteine residues near the amino-terminal end of constitutively
active Gs
or
Gq
were replaced. These
mutations prevented the G proteins from being palmitoylated or membrane bound. Loss of membrane binding abolished the ability of
Gq
to activate phospholipase C
and reduced formation of cAMP by
Gs
(34). This study was
designed to determine whether stimulation of endogenous receptors and G
proteins causes translocation in the Madin-Darby canine kidney (MDCK)
cell model of renal distal tubule. We demonstrate that bradykinin (BK)
causes translocation of G protein
-subunits of the
Gq class from the plasma membrane in whole cells and in purified plasma membrane.
 |
MATERIALS AND METHODS |
Preparation of plasma membranes.
Purified plasma membranes were prepared by aqueous two-phase partition
through a polyethylene glycol-dextran gradient as previously described
(17, 20). The yield of plasma membranes was 1-8% relative to the
initial quantity of MDCK cell crude membranes. To determine the degree of enrichment of plasma membranes, we measured
K+-stimulated, ouabain-inhibited,
p-nitrophenylphosphatase activity as
previously described (10, 20). Two-phase partition resulted in an
enrichment of 130-fold in plasma membrane marker enzyme activity.
Western blot. The purified plasma
membranes were stimulated with BK (0.1 µM), AlF (0.75 mM NaF and 5 µM AlCl3), or GTP (10 µM)
for 10 min at 37°C in a buffer containing 30 mM HEPES, pH 7.5, 100 mM NaCl, 5 mM MgCl, 3 µM GDP, 1 mM benzamidine, and 5 µg/ml soybean
trypsin inhibitor. The plasma membrane and supernatant fractions were
separated by centrifugation at 16,000 g for 15 min. In two studies, an
ultracentrifuge was used to obtain a 100,000 g pellet and supernatant fractions.
For these studies the pellet was resuspended in a volume equal to that
of the pellet (50 µl). The supernatant was subjected to SDS-PAGE and
Western blot as previously described (1) using an antiserum directed
against the carboxyl-terminal region shared by
Gq
and G11
(QLNLKEYNLV). This antibody is specific for
Gq/11
and does not crossreact
with G
i-1,
G
i-2, or
G
i-3 (data not shown). In
additional studies, G
i-1,2- and
G
i-3-specific antisera were
used to determine whether these G proteins translocated in response to
agonists. These antibodies have been previously
characterized (24). Peroxidase-conjugated goat anti-rabbit IgG (Sigma)
was used as a secondary antibody (1:10,000) and the ECL
chemiluminescent system (Amersham, Arlington Heights, IL) was used to
visualize G protein immunoreactivity. Film was exposed for 5-30
min to obtain densities in the linear range of the film. Densitometric
intensity of the control and stimulated bands was determined using a
model GS-670 imaging densitometer (Bio-Rad, Hercules, CA).
Immunofluorescence. MDCK cells plated
on glass coverslips and grown to 75% confluence in MEM media
containing 10% fetal calf serum were pretreated for 1 h with
serum-free media containing 0.5% BSA. Cells were stimulated with BK
for 10 min in a buffer containing 30 mM HEPES, pH 7.5, 100 mM NaCl, 5 mM MgCl, 1 mM benzamidine, and 5 µg/ml soybean trypsin inhibitor. The
cells were then washed twice with cold PBS, permeabilized with a buffer
containing 0.05% saponin, fixed with 3% paraformaldehyde in PBS,
quenched with 50 mM NH4Cl in PBS,
and incubated with PBS/10% FCS as described (7). Cells were then
incubated for 1 h with the
Gq/11
antibody (1:50, PBS/5%
FCS), washed six times, and then incubated for 1 h with FITC-conjugated
goat anti-rabbit IgG (Sigma). Cells were rinsed and mounted on glass
slides with vectashield (Vector Labs, Burlingame, CA). We visualized
Gq/11
immunoreactivity using a Zeis laser confocal microscope with emission at 515 nm. A
Z series was done with an image
obtained every 1 µm. Specificity of the antibody was determined by
controls utilizing preimmune serum, a monoclonal antibody against
-catenin (a component of tight junctions; Transduction Laboratories,
Lexington, KY), the Gq/11
immunogen peptide, and an irrelevant peptide (IEKFREEAEERDICIC).
 |
RESULTS |
These studies were designed to test the hypothesis that G proteins of
the Gq
family are released from
the plasma membrane in response to stimulation with BK. To establish
that activated G proteins are released directly from the plasma
membrane, rather than from some other cellular particulate fraction
such as endoplasmic reticulum or Golgi, we stimulated highly enriched
plasma membrane fractions derived from MDCK cells with BK. After
centrifugation, we performed a Western blot analysis on the supernatant
fraction to determine whether G proteins had been released from the
membrane. Exposure of plasma membranes to BK for 10 min resulted in an
increased release of Gq/11
immunoreactivity into the supernatant (Fig. 1A),
from 1.25 ± 0.56 to 2.82 ± 0.81 densitometric units
(n = 9, P < 0.05). BK caused an average
percent increase in Gq/11
immunoreactivity in the supernatant of 170 ± 53%. This
translocation of Gq/11
immunoreactivity represents a movement of G proteins out of the plasma
membranes. In additional studies, the ability of BK to induce the
translocation of other G proteins was examined. Western blots were done
on the supernatant fraction with antibodies that recognize
G
i-1,2 and
G
i-3. Unlike the results seen
with Gq/11
, no G
i-1,2 or
G
i-3 immunoreactivity was detected in the supernatant
fraction after a 10-min treatment with buffer alone. In addition, BK
did not cause the release of
G
i-1,2 or
G
i-3 immunoreactivity from the
plasma membranes (data not shown).

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Fig. 1.
A: bradykinin (BK) stimulation
increased release of Gq/11
immunoreactivity from plasma membranes. Madin-Darby canine kidney
(MDCK) cell plasma membranes were treated for 10 min with vehicle
( ) or 0.1 µM BK (+). Amount of
Gq/11 released from plasma
membrane was determined by Western blot of 16,000 g soluble fraction.
B: stimulation of BK receptor, as well
as direct stimulation of guanine nucleotide-binding regulatory proteins
(G proteins) by GTP and AlF, caused translocation of
Gq/11 immunoreactivity out of
plasma membrane at 10 min. Amount of immunoreactivity appearing in
16,000 g soluble fraction after
treatment of plasma membranes with vehicle or indicated drug was
compared for BK (n = 9), GTP
(n = 3), and AlF (n = 4).
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|
To determine whether translocation of
Gq/11
immunoreactivity could be
caused by direct activators of G proteins, we stimulated plasma
membranes with GTP or AlF (Fig. 1B).
GTP increased Gq/11
immunoreactivity in the supernatant fraction from 1.32 ± 0.22 to
3.16 ± 0.44 U (n = 3, P < 0.05),
representing an increase of 153 ± 64% in translocation. AlF
increased Gq/11
immunoreactivity in the supernatant fraction from 1.34 ± 0.15 to
2.27 ± 0.25 U (n = 4, P < 0.05), representing an increase
of 78 ± 36% in Gq/11
immunoreactivity in the soluble fraction. These data indicate that both
receptor- and nonreceptor-mediated G protein activation cause
accelerated translocation of
Gq/11
immunoreactivity out of
the plasma membrane.
To ensure that the Gq/11
immunoreactivity remaining in the supernatant was not the result of
incomplete centrifugation, we repeated these studies using an
ultracentrifuge. BK stimulation resulted in an increase in release of
Gq/11
immunoreactivity into the 100,000 g supernatant (Fig. 2). The
fraction of Gq/11
immunoreactivity appearing in the supernatant was compared with that
remaining in the pellet after centrifugation at 100,000 g for 20 min. To more accurately
determine the relative amounts of immunoreactivity in the pellet and
supernatant, we ran lanes containing equivalent amounts of supernatant
and pellet as well as lanes that contained a 1:10 dilution of the
pellet. Incubation with buffer alone resulted in translocation of 2%
of Gq/11
immunoreactivity after 10 min, whereas
incubation with BK resulted in 16% translocation of
Gq/11
immunoreactivity.

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Fig. 2.
Stimulation with BK caused an increase in release of
Gq/11 immunoreactivity from
plasma membrane and a reciprocal decrease in immunoreactivity remaining
in pellet. Western blot (A) and
densitometric analysis (B) are
shown. In these studies, plasma membranes were stimulated with 0.1 µM
BK as described, then centrifuged at 100,000 g. Lanes labeled
supernatant and
pellet 1:1 contained equivalent
fractions of soluble and particulate fractions, respectively, whereas
lanes labeled pellet 1:10 contained
only 10%. Representative of 2 experiments.
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We assessed the rapidity with which this response occurred by measuring
the amount of immunoreactivity appearing in the soluble fraction with
an increasing duration of stimulation (Fig.
3). There was a gradual increase in the
amount of translocation induced by BK over the first 10 min. The
response was maximal by 10 min and did not increase further up to 30 min. Thus BK induces a time-dependent increase in translocation of
Gq/11
immunoreactivity out of
the plasma membrane. These studies establish the possibility that Gq/11
activation leads to its
release directly from the plasma membrane. Because these studies were
done in an artificial system that lacks cytoskeletal and other membrane
support components, it is unclear whether similar translocations occur
directly from the plasma membrane in intact cells.

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Fig. 3.
Translocation of Gq/11
immunoreactivity in response to stimulation with BK is time dependent.
Amount of immunoreactivity appearing in 16,000 g soluble fraction was determined
after indicated period of stimulation with BK (0.1 µM).
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|
To determine whether translocation of
Gq/11
immunoreactivity occurred
in intact cells, as well as from purified plasma membranes, we used
immunofluorescence confocal microscopy to study the intracellular location of Gq/11
before and
after agonist treatment. We confirmed specificity of the antibody by
the failure of the preimmune serum to label G proteins and the ability
of the immunogenic peptide, but not an irrelevant peptide, to block
immunofluorescence (Fig. 4). We next
examined the ability of BK to cause translocation of
Gq/11
immunoreactivity away
from the membrane. Cross-sectional images were obtained at 1-µm
intervals from the basal to the apical surface in control and
BK-stimulated MDCK cells. Before treatment with BK,
Gq/11
immunoreactivity was
located along the basal surface of the cell near its interface with the
coverslip. A large amount of immunoreactivity could also be seen
extending up the plasma membrane at the lateral surfaces of the cell. A
small amount of immunoreactivity was seen inside the cells (Fig.
5,
A-D). This finding was
consistent with G protein localization at the cell surface, where it
could interact with both receptors and effectors. After BK treatment,
Gq/11
immunoreactivity could
still be seen at the basal and lateral surfaces, but much of the
immunoreactivity had shifted to a predominantly intracellular location
(Fig. 5, E-H). Punctate
localization of Gq/11
immunoreactivity could be seen throughout the cytoplasm. There was a
focal region of intracellular clearing that may represent exclusion of
Gq/11
from the nucleus. These
studies demonstrate, using two separate techniques, that BK stimulation
causes Gq/11
to move rapidly
from the plasma membrane to an intracellular compartment or
compartments.

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Fig. 4.
Confocal immunofluorescence micrograph of
Gq/11 and -catenin
immunoreactivity. Gq/11
antibody recognized a protein localized primarily to lateral border of
cell. Cells were treated as follows.
A: 1° antibody-preimmune rabbit
serum (1:100), 2° antibody-Oregon green-conjugated anti-rabbit
(1:200). B: 1° antibody-polyclonal
anti-Gq/11 (1:100), 2° antibody-Oregon
green-conjugated anti-rabbit (1:200). C: 1°
antibody-polyclonal anti-Gq/11
(1:100) and monoclonal anti- -catenin, 2° antibody-Oregon
green-conjugated anti-rabbit (1:200), and tetramethyl-rhodamine
isothiocyanate (TRITC)-conjugated anti-mouse (1:50).
D: 1° antibody-polyclonal
anti-Gq/11 (1:100), monoclonal
anti- -catenin, and Gq/11
immunizing peptide (1 mg/ml), 2° antibody-Oregon green-conjugated
anti-rabbit (1:200), and TRITC-conjugated anti-mouse (1:50).
E: 1° antibody-polyclonal
anti-Gq/11 (1:100), monoclonal
anti- -catenin, and Gq/11 -irrelevant
peptide (IEKFREEAEERDICIC; 1 mg/ml), 2° antibody-Oregon
green-conjugated anti-rabbit (1:200), and TRITC-conjugated anti-mouse
(1:50).
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Fig. 5.
Confocal immunofluorescence micrographs of MDCK cells treated for 10 min with vehicle (A-D) or 0.1 µM BK (E-H).
Gq/11 immunoreactivity can be
seen at basal surface in both control
(A) and stimulated
(E) cells.
B-D and
F-H represent sequential 2-µm
cuts moving apically from basal surface. In control cells, most
Gq/11 immunoreactivity is
around lateral edges of cell. After stimulation with BK, a much larger
share of Gq/11 immunoreactivity
is seen within cell. Photograph is typical of results seen in 3 separate experiments.
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 |
DISCUSSION |
This is the first direct demonstration of movement of endogenous G
proteins from the plasma membrane to another intracellular compartment.
Previous studies have shown either movement out of a crude membrane
fraction or increased appearance in whole cell low-density fractions
associated with decreased immunoreactivity in plasma membrane
fractions. The subcellular site of origin of these G proteins has not
previously been described. G proteins could be released from other
organelles present in crude membrane fractions, such as Golgi or
endoplasmic reticulum. We used both a purified fraction of plasma
membranes and direct visualization with a confocal microscope to
determine whether plasma membrane was the source of G protein
translocation. Although purified plasma membranes do contain
contaminating amounts of other membranes including Golgi, endoplasmic
reticulum, and mitochondria (17), the membranes used in the current
study showed a 130-fold increase in plasma membrane marker enzyme
activity. Therefore, it is unlikely that the magnitude of G protein
release demonstrated in this study could have come from contaminating membranes.
In the present study Gq/11
moved from the
plasma membrane to the supernatant in response to stimulation with BK.
We have demonstrated that this translocation occurs from plasma
membranes, using two techniques. Translocation occurs from
a purified preparation of plasma membranes, which lack cytoskeletal
support elements, and from the plasma membrane in whole cells, as seen
with confocal microscopy. Peak translocation by BK from plasma
membranes occurs by 10 min. In contrast to the translocation of
Gq/11
, BK did not cause
detectable translocation of Gi
subunits, although it is possible that a very small amount of
translocation occurred that was below the limit of detection.
In these studies, no additional GTP was added to the plasma membrane
preparations. Although G protein activation requires GTP binding to the
-subunit, translocation occurred without additional GTP in the
buffer. Thus either sufficient quantities of GTP must be present in the
preparation or translocation of G proteins can occur without GTP.
Concentrations of GTP have not been measured in purified plasma
membranes and may vary significantly in the vicinity of proteins with
nucleotide binding abilities. Nucleotide content has been measured
associated with the F1-ATPase
enzyme purified from the bacteria Micrococcus
lysodeikticus, in which GTP content was found to be 8.6 µM (14). These data indicate that significant concentrations of GTP
may be present in the microenvironment of the G protein, even though
the GTP concentration of the total purified plasma membrane is
miniscule. GTP content has also been measured in rod outer segments
prepared to have leaky membranes. In this preparation, which is rich in
G proteins, GTP was also present in significant amounts (27). The
concentration of GTP at the G protein in purified plasma membranes of
MDCK cells is not known, but based on these studies, it may be
significant. Thus BK and endogenous GTP may work synergistically by
interaction of an agonist-liganded BK receptor with a G protein,
followed by the binding of GTP to the G protein, to cause
translocation. These results demonstrate that either agonist binding of
the receptor or direct G protein activation causes translocation of G
proteins. Agonist binding of the receptor is not required for
translocation because the addition of AlF, which mimics the GTP-bound
state of the G protein, causes translocation.
The mechanism of translocation is unknown; however, cleavage of the
thioester bond and release of the palmitate moiety is one potential
mechanism. Palmitoylation of G proteins is required for localization to
the plasma membranes (13, 34). Palmitoylation and depalmitoylation of G
proteins is a dynamic process that is stimulated by agonists (15). A
palmitoyl protein thioesterase that cleaves palmitate has been
described (3, 4). Activation of the BK receptor could cause cleavage of
the palmitoyl-thioester linkage and release of the plasma
membrane-anchoring palmitate by one of two possible mechanisms.
Receptor activation could cause activation of the thioesterase or it
could lead to a conformational change in the G protein that would make
the thioester bond more accessible to the thioesterase. Which of these
mechanisms might be responsible for cleavage of the anchoring palmitate
is not known. It is interesting that activation by BK, which should
only affect a subset of G proteins, produced a similar magnitude of translocation to that produced by AlF, which could potentially activate
all G proteins. This suggests that G protein activation alone is not as
potent a stimulus to translocation as is receptor-mediated activation.
The role of depalmitoylation in translocation of
Gq/11
in response to
stimulation with BK has not yet been determined, but it seems a likely
mechanism in light of the known role of palmitoyl residues in membrane
targeting of Gq/11
and
Gs
.
The physiological role of translocation of G proteins away from the
plasma membrane is unclear. Translocation could be a means of
desensitization, degradation, or recycling of G proteins, or could be
important in mediating physiological responses. A decrease in the
levels of Gq/11
in the plasma
membrane might attenuate the responses mediated through those proteins.
In support of this hypothesis, long-term stimulation of GnRH or
muscarinic receptors causes a decrease in
Gq/11
immunoreactivity in
T3-1 or CHO cell extracts (18, 28, 29). In addition,
translocated G proteins could be physically associated with the
receptor and/or effectors such as phospholipase C. Movement of these
proteins out of the membrane would further attenuate the response to
stimulation. Alternatively, Gq
could be involved in a signal transduction pathway that requires it to
dissociate from the membrane. No direct role requiring dissociated G
protein
-subunits has been described, although it has been proposed
that Gs
may dissociate from the intestinal brush-border membrane in response to ADP-ribosylation by
cholera toxin and traverse to the basolateral surface where it could
activate adenylyl cyclase (6). Recently translocation of transfected
Gs
from the plasma membrane in
response to
-adrenergic receptor stimulation has been shown by
immunofluorescence microscopy. This translocation is reversed by
treatment with receptor antagonists (33). This reversibility could be
consistent with a physiological role for G protein translocation in
response to agonist stimulation. Because MDCK cells are a model for
renal distal tubule (25), basal-to-apical translocation could allow
receptor stimulation at one surface of the renal tubule to have
specific effects at other cellular sites. G protein
-subunits are
differentially expressed on different surfaces of MDCK cells (8). The
current studies document that activation of
Gq/11
results in their direct release from plasma membranes, suggesting important mechanistic implications for the function of these G proteins. Elucidation of the
function of G protein translocation will require further studies but
could serve as a mechanism for desensitization or to produce a specific
effect at other locations within the cell.
 |
ACKNOWLEDGEMENTS |
J. R. Raymond was supported by NIH Grant NS-29037, a Veterans
Affairs Merit Award, and a Grant-in-Aid from the American Heart Association. T. W. Gettys was supported by National Institute of
Diabetes and Digestive and Kidney Diseases Grant DK-42486 and a
Research Grant from the American Diabetes Association. J. M. Arthur was
the recipient of a National Kidney Foundation/American Society of
Nephrology/Sandoz research fellowship.
 |
FOOTNOTES |
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: J. M. Arthur,
Kidney Disease Program, 615 S. Preston St., Louisville, KY 40202 (E-mail: jarthur{at}kdp01.kdp-baptist.louisville.edu).
Received 7 May 1998; accepted in final form 10 December 1998.
 |
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