Departments of 1 Physiology and
2 Molecular Biology, A cDNA encoding an
Na+-glucose cotransporter type 1 (SGLT-1)-like protein was cloned from the Xenopus
laevis intestine by the 5'- and 3'-rapid
amplification of cDNA ends method. The deduced amino acid sequence was
673 residues long, with a predicted mass of 74.1 kDa and 52-53%
identity to mammalian SGLT-1s. This gene was expressed in the small
intestine and kidney, reflecting a tissue distribution similar to that
of SGLT-1. The function of the protein was studied using the
two-microelectrode voltage-clamp technique after injection of cRNA into
Xenopus laevis oocytes. Perfusion with
myo-inositol elicited about twofold
larger inward currents than perfusion with
D-glucose. The order of the
substrate specificity was myo-inositol > D-glucose > D-galactose
sodium-glucose cotransporter; sodium-myo-inositol cotransporter; Xenopus laevis oocytes; voltage
clamp
THE SODIUM-GLUCOSE cotransporter (SGLT) was first
cloned from the rabbit intestine in 1987 (9). To date, more than five SGLT clones have been isolated from various mammals, and their function
has been extensively studied by biochemical and electrophysiological methods in heterologous expression systems (mainly
Xenopus laevis oocytes) (see reviews
in Refs. 11, 36, 37). In the intestine and kidney, SGLT exists in the
apical membrane of epithelial cells facing the lumen and takes up
glucose into the cell against a glucose concentration gradient using
the Na+ electrochemical potential
difference between the inside and the outside of the cell.
Mammalian SGLTs are subdivided into the SGLT-1 and SGLT-2 types
according to their transport properties (11). Small amounts of SGLT
activity also exist in Xenopus oocytes
(34), but the properties of Xenopus SGLT are slightly different
from those of mammalian SGLTs (35). For example, neither
Xenopus SGLT nor SGLT-2 transports
galactose (24), but the transport stoichiometry of
Xenopus SGLT is 2 Na+:1 glucose, the same as that of
the mammalian SGLT-1 (35). These facts suggest that
Xenopus SGLT is, in a sense, a natural
chimera combining the properties of mammalian SGLT-1 and SGLT-2.
Therefore, to study the structure-function relationships of SGLT, we
decided to clone Xenopus SGLT and
chose the intestine as an RNA source because we hypothesized that SGLT
must be highly expressed in that tissue.
The cloned protein, which we named
Xenopus SGLT-1-like protein or
xSGLT1L,1
however, preferred myo-inositol to
glucose as a substrate when expressed in
Xenopus oocytes. Nevertheless, our
results, including amino acid sequence, transport properties, and the
expression pattern in various tissues, showed that the protein was more
similar to SGLT-1 than to the
Na+-myo-inositol
cotransporter (SMIT), another member of the SGLT cotransporter family
(32).
Cloning of xSGLT1L cDNA.
Total RNA was extracted from the Xenopus
laevis small intestine by the acid guanidinium
thiocyanate-phenol-chloroform method (3), and
poly(A)+ RNA was purified with an
mRNA purification kit (Pharmacia Biotech, Uppsala, Sweden). A 409-bp
DNA fragment was obtained by RT-PCR using the following degenerated
primers (13): sense (P1),
5'-CCTCTCTGTTTGCCAGYAATATYGGRAGTG-3'; and anti-sense (P2),
5'-GTCTGTAGKGTRTCYGTGTASATCA-3'. The PCR product was
inserted into T vector (22), which was derived from pBluescript II
SK( Preparation of cRNA.
cRNA was synthesized from a linearized plasmid containing the xSGLT1L
cDNA using T7 RNA polymerase and an mCAP mRNA capping kit (Stratagene)
according to the manufacturer's instruction manual. The transcript was
dissolved in diethyl pyrocarbonate (DEPC)-treated water at 0.5 mg/ml.
Expression of xSGLT1L in oocytes.
Xenopus oocytes were prepared as
described elsewhere (24). Briefly, a piece of the ovary from a frog was
incubated at 20°C for 1 h in the presence of 1 mg/ml of collagenase
(type I, Sigma) in Barth's solution, and, subsequently, the oocytes in
stages V-VI were defolliculated manually. The composition of
Barth's solution was 88 mM NaCl, 1 mM KCl, 2.4 mM
NaHCO3, 0.82 mM
MgSO4, 0.33 mM
Ca(NO3)2,
0.91 mM CaCl2, 10 mM HEPES-NaOH,
pH 7.4, and 0.01 mg/ml of penicillin and streptomycin. The
defolliculated oocytes were incubated overnight at 20°C in Barth's
solution, and viable oocytes were injected with 40 nl of cRNA solution
(0.5 mg/ml) or DEPC-treated water with a glass micropipette (4). The
oocytes were incubated in Barth's solution at 20°C, with daily medium changes, until use.
Electrophysiological methods.
Oocytes were subjected to electrical measurements by the
two-microelectrode voltage-clamp technique with a DPV-15D amplifier (Dia Medical System, Tokyo, Japan). Data acquisition and membrane voltage control were performed using pCLAMP version 6 software (Axon
Instruments). The intracellular voltage recording and current electrodes were filled with 3 M KCl (resistance of 1.0-1.5 M Northern blot analysis.
Ten micrograms of total RNAs from the various Xenopus
laevis tissues were electrophoresed in a
formaldehyde-agarose gel, transferred to a nylon membrane, and
subjected to hybridization. The probe, the cDNA fragment from the
Pst I site in the P2 primer region to
the 3' end of the cDNA, was labeled with
[ As shown in Fig.
1, the cDNA
for xSGLT1L was cloned and the nucleotide and deduced amino acid
sequences were determined. This open reading frame (ORF) was confirmed
by the fact that there was a stop codon (TGA) 48 nt upstream of the
first methionine of xSGLT1L in the same frame (data not shown). This
clone possessed a poly(dA) tail of only 14 nt downstream of
5'-ATTAAA-3'. On the basis of the observation that
possession of a long poly(A)+ tail
resulted in a high level of expression of cRNA in
Xenopus oocytes (19), 49 nt of
poly(dA) tail were artificially added downstream of the 3' end of
the cDNA clone. Another clone containing 80 nt of 5'-untranslated
sequence failed to be expressed into xSGLT1L in oocytes (data
not shown). In this clone, there was another initiation codon 17 nt
upstream of the first methionine of xSGLT1L (data not
shown) and this frame stopped at nt 87 (TAG). On the other hand, the
truncated clone with only 10 nt of the 5'-untranslated region was
strongly expressed into xSGLT1L in oocytes. In the course of cloning,
we obtained a short 3'-RACE product when the P4 primer was used
as the anti-sense (P4' primer in Fig.
1A) as well as the sense primer.
In the P4' primer region, 18 of 30 nt were matched, so we used the P4
primer for priming of reverse transcription in the 5'-RACE
method.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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-methyl-D-glucoside. The
current induced by myo-inositol
increased with membrane hyperpolarization and depended on external
myo-inositol and
Na+: the apparent Michaelis-Menten
constant was 0.25 ± 0.07 (SD) mM with
myo-inositol, whereas the apparent
concentration for half-maximal activation was 12.5 ± 1.0 mM and the
Hill coefficient was 1.6 ± 0.1 with
Na+. In conclusion, the cloned
protein shares features with both SGLT-1 and the
Na+-myo-inositol cotransporter.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) (Stratagene) after digestion by
EcoR V. Furthermore, extension of the
cDNA toward the 3' end was carried out using the 3' system
for rapid amplification of cDNA ends (RACE; GIBCO BRL) and nested
primer sets P3 (5'-CTGCTGGGGTCACCACTATGCCAGAGTCCC-3') and
P4 (5'-AACAAGCTCTGCAATGGGATTTGTATGTGG-3'), according to the manufacturer's instruction manual. LA
Taq DNA polymerase (Takara Shuzo,
Tokyo, Japan) was used for PCR because its fidelity is about fivefold
higher than that of the usual Taq
(personal communication from Takara Shuzo). Similarly, the 5' end
of the cDNA was obtained by the 5'-RACE method using the
following primers: P5
(5'-GCCAAGGAAGATCGGAAGTGACCGGGTCCC-3') and P6
(5'-TTCCCAGAATGAGTATTGGGAATGGCGTGG-3'). The DNA fragments made by the 5'- and 3'-RACE method were isolated and
inserted into the T vector. To eliminate PCR errors, two or more clones isolated independently were sequenced and compared, and those with a
common sequence were selected. The full-length cDNA was constructed by
ligation of the 5' and the 3' cDNAs obtained by RACE at the
Pst I site in the P2 primer region.
), and the bath electrode was a low-resistance agar bridge (28). The
oocytes were perfused with a solution containing (in mM) 88 NaCl, 2 KCl, 1.8 CaCl2, and 10 HEPES-NaOH,
pH 7.4 (24). In the Na+-free
solution, the Na+ was replaced
with choline, and the pH was adjusted with KOH in the perfusion buffer.
Voltage-clamp measurements were performed at room temperature
(20-25°C) between 3 and 18 days after the injection.
-32P]dCTP by the
random priming method using a BcaBEST
labeling kit (Takara Shuzo). Hybridization was performed at 42°C in
a solution containing 50% formamide, 5× sodium chloride-sodium
phosphate-EDTA (SSPE), 5× Denhardt's solution, and
0.5% SDS in the presence of 0.2 mg/ml of denatured salmon sperm DNA.
The membrane was washed three times for 15 min in 2×
SSPE-0.1% SDS at room temperature, then washed twice for 30 min in
0.2× SSPE-0.1% SDS at 65°C, and subjected to autoradiography.
RESULTS
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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View larger version (167K):
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Fig. 1.
A: nucleotide (top
rows, numbers on
left) and predicted amino acid
(bottom rows, numbers on
right) sequences of
Xenopus
Na+-glucose cotransporter type 1 (SGLT-1)-like protein (xSGLT1L). Primers P1-P7 are indicated by
arrows over the cDNA sequence. The 14 putative membrane-spanning
regions of the protein are indicated by boxes. * A potential
N-glycosylation site on the putative
extracellular loop. # Potential sites of phosphorylation by cAMP-
and cGMP-dependent protein kinases and protein kinase C. Polyadenylation signal is double underlined.
B: sequence alignment of xSGLT1L,
rabbit SGLT-1 (rbSGLT1), and canine
Na+-myo-inositol
cotransporter (dSMIT). Dots designate that the amino acids are the same
as those in xSGLT1L. Dashes denote gaps. Amino acids conserved in at
least 2 species are boxed. Sequence alignment and prediction of
N-glycosylation and phosphorylation
sites were performed using GENETYX version 9 (Software Development,
Tokyo, Japan).
xSGLT1L consisted of 673 amino acids (74.1 kDa), and the predicted amino acid sequence shared 53 and 46% identity with rabbit SGLT-1 (9) and canine SMIT (16), respectively (Fig. 1B). The hydrophobicity plots of the three transporters were very similar (data not shown). By analogy with the model of 14 membrane-spanning regions proposed for SGLT-1 (26, 31, 32), we identified 14 putative membrane-spanning regions in xSGLT1L and hypothesized that both termini were located outside the cell (Fig. 1A). The protein also contained potential N-glycosylation and phosphorylation sites.
Expression of xSGLT1L in cRNA-injected oocytes was examined by the
two-microelectrode voltage-clamp technique. Perfusion with myo-inositol elicited large inward
currents in cRNA-injected oocytes, but no current was observed in
control (noninjected or water-injected) oocytes (Fig.
2 and see Fig.
6A). The maximum current induced by
myo-inositol was 395 nA when oocytes
were voltage clamped at 60 mV (data not shown). The peak
amplitude of sugar-induced current in each cRNA-injected oocyte was
normalized relative to the
myo-inositol-induced current (Fig.
2C). The results indicated that the
substrate preference of xSGLT1L was
myo-inositol > D-glucose > D-galactose
-methyl-D-glucoside (
-MG).
|
The steady-state currents elicited by xSGLT1L expressed in oocytes
decreased with the membrane potential in the range from 150 to
50 mV (Fig.
3C).
When large stepwise changes of voltage were applied to cRNA-injected
oocytes, membrane currents declined exponentially to the steady state
in the absence of myo-inositol (Fig.
3A). This pre-steady-state current
was not observed in the presence of
myo-inositol (Fig.
3B) or in control (noninjected or
water-injected) oocytes (data not shown).
|
Currents elicited in cRNA-injected oocytes perfused with
myo-inositol obeyed the
Michaelis-Menten equation (Fig.
4A). The apparent Michaelis-Menten constant
(Km) for
myo-inositol was 0.25 ± 0.07 (SD)
mM (n = 7). Figure
4B shows that the currents induced by
myo-inositol at various
Na+ concentrations fitted the Hill
equation. The Hill coefficient was 1.6 ± 0.1 (n = 6), and the apparent
concentration for half-maximal activation
(K0.5) was 12.5 ± 1.0 mM. These results suggest that xSGLT1L transports two
Na+ per one
myo-inositol molecule. Phlorizin, a
competitive inhibitor of SGLT and SMIT (7, 8, 14, 19, 25),
competitively inhibited xSGLT1L (Fig.
4C), and the apparent inhibition
constant (Ki)
was 7.3 ± 2.3 µM (n = 7).
|
xSGLT1L also showed Michaelis-Menten-type kinetics with external D-glucose. The apparent Km was 6.3 ± 1.2 (SD) mM, as determined in five xSGLT1L-expressing oocytes from two batches (data not shown). This value was ~25-fold greater than that with myo-inositol.
It is reported that SGLT-1 works as a
Na+ uniporter in the absence of
sugar and generates an inward current (20, 33). This is called the
Na+ leak; it represents ~20%
(or less) of the maximum sugar-induced current and is inhibited by
phlorizin, like the sugar-induced current. We therefore perfused the
oocytes expressing xSGLT1L with 0.5 mM phlorizin. The baseline current,
however, was not changed, or was changed very little, by this treatment
(Fig. 5).
|
To evaluate the physiological function of xSGLT1L, we injected
poly(A)+ RNA derived from the
Xenopus laevis small intestine into
oocytes and measured the membrane currents. In control (noninjected)
oocytes, L-alanine,
D-glucose, and -MG induced
small inward currents, whereas
myo-inositol did not (Fig.
6, A and
C). The
D-glucose and
-MG responses
were probably due to the endogenous SGLT in Xenopus oocytes (24, 34, 35), and the
L-alanine response might have
been caused by the endogenous amino acid transporter (5). After the
poly(A)+ RNA injection, the
responses elicited by L-alanine
and D-glucose became larger than
the responses in the control oocytes (Fig. 6,
B and
C), but
myo-inositol induced no response in
RNA-injected oocytes.
|
Northern blot analysis revealed that an xSGLT1L mRNA of ~3.3 kb was
abundantly expressed in the small intestine and kidney (Fig.
7).
|
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DISCUSSION |
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The amino acid sequence of xSGLT1L shared 52-53% identity with those of human (10), rabbit (9), and rat (17) SGLT-1s, 50% identity with that of human SGLT-2 (14), and 45-46% identity with those of human (2), canine (16), and bovine (21) SMITs. Many amino acids important for the SGLT-1 function were conserved in xSGLT1L, including Asn-246, a potential N-glycosylation site, and Arg-421, which is thought to be involved in trafficking SGLT-1 to the plasma membrane (18). In addition, 15 of 19 sites that were point mutated in SGLT-1 from patients suffering from glucose-galactose malabsorption (23) were also conserved: the sites were Asp-27, Arg-134, Ser-158, Ala-165, Trp-270, Cys-286, Gln-289, Arg-294, Ala-298, Arg-373, Ala-382, Phe-399, Gly-420, Val-464, and Arg-493.
However, xSGLT1L preferred
myo-inositol to
D-glucose as a substrate
when expressed in Xenopus oocytes, and
it showed very low activities toward
D-galactose and -MG, <12%
of that demonstrated toward
myo-inositol.
-MG is well known as
a specific substrate for SGLT but not for facilitated glucose
transporters (11, 14, 19, 36). Therefore, we named the transporter
cloned in this study Xenopus
SGLT-1-like protein or xSGLT1L. Its substrate specificity was more
similar to that of SMIT (7) than to the specificities of SGLT-1 (7) and
SGLT-2 (14, 19). Hereafter, we discuss points of similarity and
difference among xSGLT1L, SGLT-1, and SMIT (Table
1).
|
xSGLT1L transports two Na+ per one
organic solute, as do SMIT (7) and SGLT-1 (11, 12, 36), whereas SGLT-2
transports one Na+ per
D-glucose (14, 19). The
K0.5 value of
xSGLT1L for Na+ is ~13 mM at a
membrane potential of 60 mV, which is similar to that of SGLT-1
(5-15 mM) (12) but not to that of SMIT (75 mM at
50 mV)
(7). xSGLT1L shares two additional properties with SGLT-1 and SMIT:
Michaelis-Menten-type kinetics for the organic substrate, with
Km = 0.05-0.5 mM, and competitive inhibition by phlorizin (7, 12).
These common features may be due to the sequence homology among these
three transporters, especially in the
NH2-terminal half. On the other
hand, their different substrate specificities may be explained by the
difference in the COOH-terminal half, where the homology is relatively
low. This is consistent with the recent report that the last five
transmembrane helixes form the sugar pathway through SGLT-1 (26).
In the absence of myo-inositol, xSGLT1L showed a pre-steady-state current after stepwise changes of the membrane potential (Fig. 3A). Similar results were observed for SGLT-1 and SMIT expressed in Xenopus oocytes, and the current has been attributed to the charge movements due to Na+ binding-dissociation and a conformational change of the transporters (7, 8, 25). This fact also suggests functional similarity between xSGLT1L and mammalian homologs.
SGLT-1 generates the so-called Na+ leak, which is an inward current carried by Na+ in the absence of sugar (20, 33), whereas SMIT does not (7). Because the leak is inhibited by phlorizin, the data in Fig. 5 imply that xSGLT1L produces negligible Na+ leak, if any. In this respect, xSGLT1L resembles SMIT rather than SGLT-1.
Northern blot analysis showed that xSGLT1L mRNA was expressed in the small intestine and kidney. The size of the mRNA was estimated at ~3.3 kb, which was ~0.8 kb longer than our cDNA clone. This difference was probably due to a long poly(A)+ tail of 0.6 kb or more, which was present in the mRNA but missing from our cDNA clone. In addition, the 5'-untranslated region of the 3.3-kb xSGLT1L mRNA may be relatively long because one cDNA clone derived from 5'-RACE had a 5'-untranslated region of ~190 bp (data not shown).
SGLT-1 is strongly expressed in the small intestine, at a much lower level in the kidney, and very weakly in the lung and liver (11). In addition, the protein is expressed in the brain (27). On the other hand, SMIT is expressed in various tissues, such as kidney, brain, placenta, pancreas, heart, skeletal muscle, and lung (2), but not in the ileal mucosa (16). Accordingly, the main difference between SGLT-1 and SMIT is expression in the small intestine. The Northern analysis shown in Fig. 7, therefore, indicates that the expression pattern of xSGLT1L is more similar to that of SGLT-1 than to that of SMIT.
SGLT-1 and SMIT also differ in terms of the structure of their genomic DNAs. The coding region of SMIT mRNA resides within one exon (2, 29), whereas SGLT-1 mRNA is composed of 15 exons (30). It is thought that the SMIT gene might have been retrotransposed from the SGLT-1 gene (21, 29). In preliminary work, we performed PCR on the Xenopus laevis genome using primers P4-P7 (Fig. 1A), and the results suggested that the region between P4 and P6 could not be mapped into one exon (data not shown). This fact strengthened our conclusion that the cloned protein is not SMIT but is a member of the SGLT-1 subfamily. Therefore, investigating the genomic structure of xSGLT1L should shed light on the evolution of the SGLT family.
xSGLT1L appears to be different from the endogenous SGLT in
Xenopus oocytes (24, 34, 35), the
transporter we first aimed to clone, because expression of xSGLT1L was
not observed in the ovary with the use of Northern analysis (Fig.
7C). Furthermore, noninjected
oocytes occasionally showed small inward currents when
D-glucose or -MG was applied,
but no response was elicited by
myo-inositol (Fig.
6A). This fact implies that the
inward current was not due to xSGLT1L but rather to the endogenous SGLT.
Does xSGLT1L play a role in nutrient absorption, like SGLT-1, in the
small intestine? To address this question, we recorded membrane
currents from oocytes injected with
poly(A)+ RNA purified from the
small intestine of Xenopus laevis.
However, the poly(A)+ RNA-injected
oocytes produced no current when perfused with
myo-inositol. On the other hand, the
response induced by D-glucose or
L-alanine was significantly
increased, which suggested the expression of their transporters in the
small intestine. It is still unclear why the expression of xSGLT1L was
not observed. However, as noted above, there is another initiation
codon 17 nt upstream of the first methionine of xSGLT1L, and the
protein was not expressed in oocytes injected with cRNA containing this
region. Because this ATG resides within the sequence
5'-TGGCA-3',
it probably does not function as an initiation codon (15); however,
translation of xSGLT1L might be repressed by this sequence in oocytes.
Alternatively, xSGLT1L might have been derived from
Xenopus SGLT mRNA by a mutation (or
mutations) that occurred during RACE cloning, and, as a result, the
substrate specificity of the SGLT might have been drastically changed.
Although we confirmed the sequence of the open reading frame (ORF)
using at least two independent clones (in 25% of the ORF by using two
and in 75% of the ORF by using three or more independent clones), we
still might not have been able to detect a mutation (or mutations),
especially if it occurred in an early cycle of PCR. However, the
production of simultaneous changes in the substrate specificity and the
Na+ leak by a mutation (or
mutations) is unlikely, so xSGLT1L cDNA is very unlikely to be a PCR
artifact. More experiments are needed to elucidate the physiological
function of xSGLT1L.
In conclusion, the protein cloned in this study shares properties with both SGLT-1 and SMIT: whereas the substrate specificity and the absence of the Na+ leak resemble the properties of SMIT, other kinetic parameters, the tissue distribution of transcription and the genomic organization, indicate a close resemblance to SGLT-1.
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ACKNOWLEDGEMENTS |
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We thank Dr. Osamu Ichikawa for critical comments on the manuscript and Kohichi Adachi for excellent technical assistance.
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FOOTNOTES |
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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.
1 The nucleotide sequence reported in this paper has been deposited in the DDBJ-EMBL-GenBank databases with the accession no. AB008225.
Address for reprint requests and other correspondence: K. Nagata, Dept. of Physiology, Faculty of Medicine, Tottori Univ., Nishimachi 86, Yonago 683-8503, Japan (E-mail: physiol{at}grape.med.tottori-u.ac.jp).
Received 20 March 1998; accepted in final form 5 February 1999.
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