1 Department of Nutrition, School of Medicine, The University of Tokushima, Tokushima City, Tokushima 770-8503; and 2 Gastrointestinal Unit, Kyoto Red Cross Hospital, Kyoto City, Kyoto 605-0981, Japan
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ABSTRACT |
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We previously
reported that primary cultures of guinea pig gastric pit cells
expressed all of the phagocyte NADPH oxidase components (gp91-, p22-,
p67-, p47-, and p40-phox) and could spontaneously release
superoxide anion (O2). We demonstrate here that pit
cells express a nonphagocyte-specific gp91-phox homolog
(Mox1) but not gp91-phox. Inclusion of catalase significantly inhibited [3H]thymidine uptake during the
initial 2 days of culture. Pit cells, matured on day 2,
slowly underwent spontaneous apoptosis. Scavenging O2
and related oxidants by superoxide dismutase plus catalase or N-acetyl cysteine (NAC) and inhibiting Mox1 oxidase by
diphenylene iodonium activated caspase 3-like proteases and markedly
enhanced chromatin condensation and DNA fragmentation. This accelerated apoptosis was completely blocked by a caspase inhibitor,
z-Val-Ala-Asp-CH2F. Mox1-derived reactive oxygen
intermediates constitutively activated nuclear factor-
B, and
inhibition of this activity by nuclear factor-
B decoy
oligodeoxynucleotide accelerated their spontaneous apoptosis. These
results suggest that O2
produced by the pit cell Mox1
oxidase may play a crucial role in the regulation of their
spontaneous apoptosis as well as cell proliferation.
NADPH oxidase; superoxide anion; hydrogen peroxide; antiapoptosis; cell growth
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INTRODUCTION |
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PHAGOCYTE NADPH
OXIDASE-DERIVED superoxide anion (O2) and
related oxygen metabolites play a crucial role in microbial and tumoricidal activities (1, 27). These oxygen metabolites are now considered to be an important signal for initiation of inflammatory and immune responses. The phagocyte NADPH oxidase consists
of a membrane-bound cytochrome b558 heterodimer
(gp91-phox and p22-phox) and three cytosolic
components (p67-phox, p47-phox, and
p40-phox) and is modulated by Rac1/2 (see Ref.
1 for review). On activation, the cytosolic components
translocate to the plasma membrane, where the functional NADPH oxidase
is assembled.
There is increasing evidence that distinct types of nonphagocytic
cells, such as vascular smooth muscle cells (18),
glomerular mesangial cells (12), endothelial cells
(13), and fibroblasts (15), express p22-,
p67-, and p47-phox and can produce small amounts of
O2. These cells were suggested to have a low
potential cytochrome b in their plasma membranes whose
spectroscopic characteristics were similar to those of the phagocyte
cytochrome b558 (12). Furthermore,
Meier et al. (15) showed that fibroblasts isolated from a
patient with X-linked chronic granulomatous disease causing genetic
defects in gp91-phox are able to generate
O2
similarly to fibroblasts from a healthy donor. On
the basis of these observations, it has been proposed that a
genetically distinct isoenzyme of gp91-phox must exist in
the nonphagocytic cells having O2
-releasing activity
(12). Recently, Suh et al. (28) molecularly identified a novel gp91-phox isoenzyme, termed a mitogenic
oxidase (Mox1). They also demonstrated that Mox1-derived
O2
regulated serum-dependent growth of aortic
vascular smooth muscle cells and that overexpression of Mox1 in NIH-3T3
cells enhanced O2
production and induced abnormal
cell growth and transformation (28).
We previously reported that primary cultures of guinea pig gastric pit
cells express all of the essential components of the phagocyte NADPH
oxidase and can release abundant O2 through the
oxidase (30, 31). However, the pit cell oxidase displays
features different from those of the phagocyte oxidase. Pit cells in
culture spontaneously and continuously produce O2
for
up to several days and do not exhibit respiratory burst-like production. Potent stimulators for the phagocyte NADPH oxidase, such as
phorbol diester, do not increase the rate of O2
production by pit cells (30). O2
generated by gastric pit cells is not lethal to the cells themselves, as similarly observed in endothelial cells (13) and
vascular smooth muscle cells (18). However, the
physiological implications of O2
production by
gastric pit cells remain to be elucidated.
We demonstrate here that guinea pig gastric pit cells express Mox1 but
not gp91-phox. Furthermore, we suggest that
O2 derived from the Mox1 oxidase may regulate
spontaneous apoptosis of pit cells through modulating the caspase- and
nuclear factor-
B (NF-
B)-dependent pathways.
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MATERIALS AND METHODS |
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Preparation and culture of gastric mucosal cells.
All procedures involving animals were approved by the Animal Care and
Use Committee of the University of Tokushima. Male guinea pigs,
weighing ~250 g, were purchased from Shizuoka Laboratory Animal
Center (Shizuoka, Japan). Gastric mucosal cells were isolated aseptically from guinea pig fundic glands and cultured in RPMI 1640, supplemented with 10% FCS, 2 mM glutamine, 100 U/ml
penicillin, and 50 µg/ml gentamicin, as previously described
(20, 21, 23, 30). The cell populations were
determined by cytochemical and immunocytochemical analyses as well as
transmission electron microscopic examination, as previously described
(24, 30). The cultured cells consisted of pit cells
(~90%), parietal cells (5%), mucous neck cells (<1%), fibroblasts
(<1%), and pre-pit cells (5%). Among these cell populations, only
mature pit cells expressed detectable levels of p47-phox and
p67-phox, and their O2 production was
confirmed by nitroblue tetrazolium staining (30). Cell
growth was estimated by counting cell number and by measuring the
[3H]thymidine uptake, as previously described
(17).
Preparation of guinea pig neutrophils. Guinea pig peritoneal neutrophils were obtained by flushing the peritoneal cavity with saline 12 h after an intraperitoneal injection of 25 ml of 3% thioglycolate broth as described previously (30). Microscopic observation after Giemsa staining revealed that >90% of peritoneal exudate cells were neutrophils.
Measurement of O2 release from gastric pit
cells.
O2
release from gastric pit cells was assayed by
measuring the superoxide dismutase (SOD)-inhibitable reduction of
ferricytochrome c as described previously (30).
The reduction of ferricytochrome c was
spectrophotometrically determined at 550 nm, and the amount of
O2
release was expressed as nanomoles per milligram
of protein per hour.
Analysis of DNA fragmentation. Cells were washed with PBS and then lysed by treatment for 30 min at 4°C with lysis buffer consisting of 10 mM Tris · HCl (pH 8.0), 10 mM EDTA, and 0.5% Triton X-100. Intact chromatin DNA was pelleted by centrifugation of the lysate at 20,000 g for 30 min at 4°C. The amounts of intact chromatin DNA in the pellets and soluble, fragmented DNA in the supernatants were spectrophotometrically determined at 600 nm by using the diphenylamine reagent (5). Percentage of fragmented DNA recovered in the supernatant was calculated.
The DNA in the supernatant was extracted twice with an equal volume of phenol-chloroform mixture (1:1) and then precipitated with 0.1 volume of 3 M sodium acetate (pH 5.2) and 2 volumes of ethanol. After treatment with 40 µg/ml of RNase A for 1 h at 37°C and then with 50 µg/ml pronase K for 30 min at 37°C, the extracted DNA was subjected to 2% agarose gel electrophoresis to detect nucleosome-sized DNA ladders. The fragmented DNA was stained with ethidium bromide and visualized by ultraviolet transillumination.Assessment of nuclear morphology. Chromosomal condensation and fragmentation were assessed by staining with the fluorescent dye Hoechst 33342. Cells were fixed with 2% paraformaldehyde in PBS for 30 min at room temperature. After fixation, they were washed twice with PBS and then exposed to 5 µg/ml of Hoechst 33342 in PBS for 30 min at room temperature. After being washed, samples were mounted with glycerol-PBS (1:1 vol/vol) and subjected to fluorescence microscopic examination.
Measurement of caspase 3-like activity. After cells were washed with ice-cold PBS, they were lysed with 100 mM HEPES buffer (pH 7.5), containing 10% sucrose, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml pepstatin, and left on ice for 30 min. After centrifugation at 15,000 g for 30 min at 4°C, the supernatant (100 µg protein) was mixed with 150 µl of 100 mM HEPES buffer (pH 7.5), containing 10% sucrose, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml pepstatin, and 400 µM of the synthetic substrate N-acetyl-Asp-Glu-Val-Asp-p-nitroanilide (BIOMOL Research Laboratories, Plymouth, PA). The mixture was incubated at 37°C for 1 h. Caspase 3-like activity was assayed by measuring the increase in absorbance at 405 nm. The reaction mixture lacking N-acetyl-Asp-Glu-Val-Asp-p-nitroanilide or cellular proteins was used as a negative control. Enzyme activity was expressed as increase in the absorbance per milligram of protein per hour.
Decoy oligodeoxynucleotide experiments.
Double-stranded NF-B decoy and mutant decoy oligodeoxynucleotides
(ODNs) were prepared from the complementary single-stranded phosphorothioate-bonded ODN by annealing. The sequence of each decoy
ODN was as follows: NF-
B decoy, 5'-AGTTGAGGGGACTTTCCCAGGC-3'; mutant
decoy, 5'-AGTTGAGCTCACTTTATCAGGC-3'. One nanomole of double-stranded decoy ODN was mixed with 5 µg of LipofectAMINE (Life Technologies, Rockville, MD) in 0.1 ml of serum-free OPTI-MEM (Life Technologies), and then the mixture was incubated for 20 min at room temperature to
form liposome complexes. After 0.9 ml of serum-free OPTI-MEM was added
to the mixture, cultured gastric mucosal cells were incubated with the
liposome complexes for 8 h at 37°C. Thereafter, the decoy
ODN-containing medium was replaced by fresh serum-free RPMI 1640, and
cells were further incubated for 12 h to assess their apoptosis.
Gel mobility shift assay.
Nuclear proteins were prepared from cultured gastric mucosal cells, and
the activation of NF-B was examined by gel mobility shift assay, as
described previously (20, 23).
RT-PCR and Northern blot analyses. Total RNA was isolated with an acid guanidinium thiocyanate-phenol-chloroform mixture (6). RT reaction and PCR were performed using a TaKaRa RT-PCR kit (TaKaRa, Tokyo, Japan). The sequences of primers used were as follows: human gp91-phox, 5'-CATCATCTCTTTGTGATCTTCT-3' (sense) and 5'-CTTAGGTAGTTTCCACGCATC-3' (antisense) (25); human mox1, 5'-GCTCATTTTGCAGCCGCA-3' (sense) and 5'-AGAATGACCGGTGCAAGG-3' (antisense) (28). Amplified products were separated in a 6% polyacrylamide gel, stained with ethidium bromide, and visualized by ultraviolet transillumination. The resultant PCR products were purified from the gel, ligated into pCR2.1-TOPO vector (Invitrogen, Carlsbad, CA), and transformed into JM109 cells. Transformed plasmids containing the appropriate insert DNA were selected and sequenced with a DNA sequencer (model ABI 373; PE Biosystems Japan, Tokyo, Japan).
The amplified 589-bp cDNA for guinea pig mox1 described above was used as a probe for Northern blotting. The cDNA probe for guinea pig mox1 or human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was prelabeled with [Statistical analysis. ANOVA and Scheffé's test were used to determine statistically significant differences. Differences were considered significant if P < 0.05.
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RESULTS |
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Expression of Mox1 transcript in gastric mucosal cells. In a previous report (31), we showed that cultured guinea pig gastric mucosal cells had immunoreactive proteins to an antibody raised against synthetic peptide corresponding to human gp91-phox (residues 508-526). However, the molecular masses of the pit cell gp91-phox (51-53 kDa) were somewhat different from those of the neutrophil gp91-phox (51-66 kDa) (31). The amino acid sequence of the epitope of human gp91-phox is 48% identical to that of human Mox1 (28); therefore, it is possible that the antibody recognized the Mox1 but not gp91-phox.
To address this issue, primer sets were designed to specifically hybridize the gp91-phox or mox1 mRNA, respectively. RT-PCR with the primer set for gp91-phox did not amplify any products in gastric mucosal cells (Fig. 1A), whereas RT-PCR for mox1 resulted in the generation of a single product corresponding to the predicted base pair size for the mox1 transcript in gastric mucosal cells, as well as Caco-2 cells, which were reported to express the mox1 mRNA (28). The mox1 transcript could not be detected in guinea pig neutrophils. The amplified PCR product from gastric mucosal cells was confirmed to be a guinea pig mox1 by DNA sequencing; the sequence of the product showed 84% identity to the human mox1 cDNA (454-1042 bp; data not shown). Furthermore, Northern blot analysis with the PCR product as a probe showed that gastric mucosal cells expressed a significant amount of 5-kb mox1 mRNA that was not detected in neutrophils (Fig. 1B).
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Effects of antioxidants on growth and death of gastric mucosal
cells.
To reveal the physiological role of O2 in gastric
mucosal cells, we at first examined the relationship between
O2
-producing capacity and cell growth (Fig.
2). After starting culture, isolated
cells attached to culture plates within several hours and began to
grow, with a doubling time of ~24 h (17). As shown in
Fig. 2A, O2
-producing activity was
observed in the cells that were already attached and grown on the
culture plates within 24 h. The activity continued to increase and
reached a maximum at 48 h when growing cells became confluent.
Since O2
-producing activity appeared to be associated
with growth of gastric mucosal cells, we examined the effects of SOD
and/or catalase on the cell growth. Inclusion of SOD did not change the
[3H]thymidine uptake, but catalase or catalase plus SOD
significantly suppressed the uptake during the proliferation periods
(36-48 h after cultivation) by 50 or 45%, respectively (Fig.
2B), suggesting that hydrogen peroxide dismutated from
O2
might function as a signal molecule for cell
proliferation.
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Effects of antioxidants on apoptosis.
The decline of O2 production by mature pit cells
seemed to be related to spontaneous apoptotic cell death, and
externally supplemented hydrogen peroxide significantly inhibited their
death, leading us to consider that the Mox1 oxidase might regulate the cell death. To confirm this, we examined whether scavenging of O2
and its related oxygen intermediates affected the
cell viability. FCS could stimulate proliferation of small
numbers of immature cells (<5%) to compensate for the cell loss
caused by apoptosis. To correctly evaluate the apoptotic cell
loss, cells cultured for 48 h were incubated in FCS-free RPMI 1640 supplemented with saline as a vehicle, SOD plus catalase, or
N-acetyl cysteine (NAC) for the indicated hours, and cell
viability was monitored by trypan blue exclusion test (Fig.
3). Vehicle-treated cells slowly lost their viability, and ~6% of the cells were dead at 16 h (Fig. 3A). On the other hand, inclusion of SOD plus catalase or
NAC significantly accelerated the death, and 10% of the cells died at
8 h. Percentage of DNA ladder increased in proportion with the
increase in number of dead cells in the presence of SOD plus catalase
or NAC (Fig. 3B).
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Effects of antioxidants on caspase 3-like activity.
We examined the mechanism by which Mox1-derived reactive oxygen
intermediates (ROI) suppressed pit cell apoptosis. As shown in Fig.
5A, a general caspase
inhibitor, z-Val-Ala-Asp-CH2F (z-VAD-fmk), completely
blocked the apoptotic DNA fragmentation induced by the antioxidants,
suggesting that O2 and/or hydrogen peroxide may
interfere with the caspase-dependent apoptosis pathway. To confirm
this, we measured caspase 3-like activity by calorimetric assay. In
vehicle-treated cells, caspase 3-like activity slightly increased at
8 h (Fig. 5B). SOD plus catalase or NAC significantly
increased the activity at 4 h before the appearance of DNA
fragmentation (Fig. 4D). Furthermore, when O2
production was inhibited by diphenylene iodonium,
a potent inhibitor for the pit cell Mox1 oxidase (30), the
caspase 3-like activity was further increased (Fig. 5B).
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NF-B activation.
Gastric pit cells possess oxidant-sensitive transcription factor
NF-
B (23), which plays a critical role in protection
from apoptosis in many types of cells (7, 32). Gel
mobility shift assay showed that untreated cells growing in RPMI 1640 supplemented with 10% FCS constitutively contained low levels of
NF-
B binding activity (Fig. 6). When
cells were incubated in FCS-free RPMI 1640 supplemented with vehicle
for 2 h, this binding activity was pronounced. This NF-
B
activation was almost completely blocked by inclusion of SOD plus
catalase or NAC, suggesting that production of O2
and
hydrogen peroxide by pit cells in FCS-free conditions might stimulate
NF-
B-dependent antiapoptotic pathways.
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DISCUSSION |
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Our previous studies (30, 31) showed that the
O2-generating system in gastric pit cells shared
functional and structural similarities with the phagocyte NADPH oxidase
by cell-free reconstitution experiments and immunoblot analysis with
antibodies against the human neutrophil phox components
(gp91-, p22-, p67-, p47-, and p40-phox). After our reports,
a nonphagocyte-specific isoenzyme of gp91-phox (Mox1) was
molecularly identified (28). The human Mox1 protein
consists of 564 amino acids and has 56% sequence identity to the 569 residues for human gp91-phox. The flavin and pyridine
nucleotide binding sites are conserved between gp91-phox and
Mox1. However, Mox1 lacks asparagine-linked glycosylation sites that
are present in gp91-phox (25, 28). This may be the reason why Mox1 was detected as an immunoreactive protein to the
anti-gp91-phox antibody with a somewhat smaller molecular mass than that of gp91-phox (31). In this
study, we could confirm that gastric pit cells express the transcript
of mox1, but not gp91-phox, by DNA sequencing the
RT-PCR product and by Northern blot analysis.
We provide here a possible physiological role of Mox1-derived ROI in
gastric pit cells. The activity of the pit cell Mox1 oxidase increased
in association with their proliferation. [3H]thymidine
uptake experiments suggested that hydrogen peroxide, possibly
dismutated from the Mox1 oxidase-derived O2, might
stimulate pit cell growth during the proliferation period, as in
fibroblasts (16) and vascular smooth muscle cells
(28). Furthermore, the Mox1-derived ROI modulated
spontaneous apoptosis of mature pit cells. After being cultured for
48 h, ~90% of the cells matured into final differentiated cells
having large secretory granules in the ectoplasm, which is
characteristic of mature pit cells or surface mucous cells
(24). The Mox1 oxidase activity of these cells gradually
reduced in association with the progression of spontaneous apoptosis.
During this stage, treatment of cells with a low concentration of
hydrogen peroxide partially suppressed their spontaneous apoptosis.
Alternatively, scavenging O2
and related oxidants
significantly accelerated their apoptosis. These findings suggest that
O2
produced by pit cells may not only stimulate cell
growth but may also suppress spontaneous apoptosis of mature pit cells.
There is growing evidence that ROI are key molecules for regulation of
apoptosis and survival. Exposure of cells to sublethal doses of ROI,
such as O2
and hydrogen peroxide, is known to exert
stimulatory effects on their proliferation (3, 4, 29) and
to induce tolerance against several apoptogenic stimuli (2,
14). Furthermore, several types of cells, particularly tumor
cells, spontaneously produce O2
to escape from
apoptosis induced by NO (14) and Fas (8).
We also provide a possible mechanism(s) for antiapoptotic actions of
Mox1-derived ROI in gastric pit cells. The DNA fragmentation initiated
by scavenging ROI was completely abolished by z-VAD-fmk. On the other
hand, removal of ROI or inhibition of the Mox1 oxidase activity
significantly increased caspase 3-like activity, suggesting that
Mox1-derived ROI could interfere with the apoptotic processes of
activation of group II caspases (caspases 2, 3, and 7). These caspases
are cysteine-dependent proteases and sensitive to the cellular
reduction/oxidation status. It has been reported that ROI, including
O2, can inhibit the activation of caspase 3-like
enzymes and also directly inactivate them (11, 19).
ROI produced by the Mox1 oxidase could constitutively activate NF-B
in gastric mucosal cells, which may be an important signal for
proliferation and/or survival. In fact, the
O2
-dependent activation of NF-
B was shown to
attenuate nitric oxide- and tumor necrosis factor-
-induced apoptosis
(10, 14). In addition, NF-
B is now known to upregulate
transcription of several antiapoptotic genes, including cellular
inhibitor of apoptosis (cIAP2) (7), Bcl-2 homolog Bfl-1/A1
(32), and cyclooxygenase-2 (14). The present
experiments did not identify the antiapoptotic genes downstream of
NF-
B activation in gastric pit cells. However, the activation of
NF-
B by Mox1-derived ROI appeared to play a crucial role in the
suppression of spontaneous apoptosis of pit cells, since inhibition of
NF-
B-binding activity by NF-
B decoy ODN significantly enhanced
their apoptosis, and NAC, a potent inhibitor of NF-
B activation
(26), could induce apoptosis, similarly to the way that
SOD plus catalase did.
To our knowledge, this is the first time that gastric pit cells in
culture have been shown to express a potent Mox1 oxidase, and we
suggest that O2 derived from the oxidase acts as an
important signal for proliferation and survival of the cells.
Considering the close relationship between the activity of the pit cell
Mox1 oxidase and mitogenic activity or death, the strictly regulated
production of O2
may directly or indirectly determine
whether pit cells proliferate, inhibit growth, or enter the apoptotic
pathway, at least partially in vitro. The ROI-dependent regulation of
proliferation and cell death is known to depend on the concentration
and the flux rate of its formation (9). If their
O2
production is dysregulated, the balance between
cell growth and death may be damaged. In fact, overexpression of Mox1
in NIH-3T3 cells enhanced O2
-producing ability,
resulting in abnormal cell growth and transformation in vitro and
aggressive growth of the implanted tumor cells in vivo
(28). In addition to the potent mitogenic activity, tumor cells harboring O2
-producing ability show a
resistance to apoptosis induced by Fas, tumor necrosis factor-
, and
anticancer drugs (8, 10, 19).
We already examined the expression of the Mox1 oxidase components in
human gastric glands and found that pit cells, but not other types of
gastric epithelial cells, expressed immunoreactive materials to
antibodies against p47-phox and p67-phox and to
an antibody recognizing both gp91-phox and Mox1
(22). At present, we do not know whether the Mox1 oxidase
regulates rapid proliferation of a pit cell lineage and continuous cell
loss by apoptosis in vivo. To reveal the physiological meaning of the
O2-generating system in vivo, more detailed
examinations of the expression of the Mox1 oxidase in human gastric
glands are needed.
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ACKNOWLEDGEMENTS |
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A part of this work was supported by a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Science and Culture (to S. Teshima and K. Rokutan).
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FOOTNOTES |
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Address for reprint requests and other correspondence: K. Rokutan, Dept. of Nutrition, School of Medicine, The Univ. of Tokushima, 3-18-15 Kuramoto-cho, Tokushima City, Tokushima 770-8503, Japan (E-mail: rokutan{at}nutr.med.tokushima-u.ac.jp).
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 17 March 2000; accepted in final form 12 July 2000.
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