Department of Molecular Biology, Yokohama City University School of Medicine, Kanazawa-Ku, Yokohama 236, Japan
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ABSTRACT |
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Protein kinase C (PKC) has been reported to
be associated with the activation of extracellular signal-regulated
kinase (ERK) by hyperosmolality. However, it is unclear whether
hyperosmolality induces PKC activation and which PKC isoforms are
involved in ERK activation. In this study, we demonstrate that NaCl
increases total PKC activity and induces PKC, PKC
, and
PKC
translocation from the cytosol to the membrane in
NIH/3T3 cells, suggesting that hyperosmotic stress activates
conventional PKC (cPKC) and novel PKC (nPKC). Further studies show that
NaCl-inducible ERK1 and ERK2 (ERK1/2) activation is a consequence of
cPKC and nPKC activation, because either downregulation with
12-O-tetradecanoylphorbol 13-acetate
or selective inhibition of cPKC and nPKC by GF-109203X and rottlerin
largely inhibited the stimulation of ERK1/2 phosphorylation by NaCl. In
addition, we show that NaCl increases diacylglycerol (DAG) levels and
that a phospholipase C (PLC) inhibitor, U-73122, inhibits NaCl-induced
ERK1/2 phosphorylation. These results, together, suggest that a
hyperosmotic NaCl-induced signaling pathway that leads to activation of
ERK1/2 may sequentially involve PLC activation, DAG release, and cPKC
and nPKC activation.
sodium chloride; phospholipase C; diacylglycerol; phosphorylation; novel and conventional protein kinase C; extracellular signal-regulated kinase-1 and -2
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INTRODUCTION |
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CELLS IN THE RENAL MEDULLA are normally exposed to an extremely hyperosmotic milieu caused by accumulation of high levels of NaCl and urea during the process of urinary concentration (4). A change in osmolality due to cellular hydration under the influence of hormones, nutrients, and oxidative stress has been observed in all other cell types studied so far (17). The cells react to increased osmolality with activation of ion transport in the plasma membrane, alterations in metabolic processes, and induction of gene transcription and protein phosphorylation (17). Over the past two decades, the regulation of these events in response to osmotic changes had become much more thoroughly understood. However, it is largely unknown which intracellular signals activate or control these osmolality-regulatory responses and, especially, regulate gene expression. It has been proposed that transcription factors might be involved in a coordinated program of gene expression governing adaptation to hyperosmotic stress (9). Recently, Cohen and Gullans (9) demonstrated that high concentrations of urea and NaCl can increase expression of two immediate-early gene transcription factors, c-Fos and Egr-1, in tissues of renal cells [Madin-Darby canine kidney (MDCK) and LLC-PK1]. Expression of c-Fos was also detectable in nasal gland tissue from ducklings and neuronal tissue from rats in response to hyperosmotic stimulation (13, 18, 32).
Because the activation of transcription factors is usually regulated by distinct signaling pathways, some recent studies have focused on the identification of signaling molecules involved in the osmotic regulation of transcription factor expression. In renal inner medullary collecting duct cells (mIMCD3), it has been shown that hyperosmotic urea-induced transcription of Egr-1 is mediated by extracellular signal-regulated kinase (ERK) (8). Hypotonicity-mediated transcriptional regulation of this gene is also partially involved in ERK activation in this cell type (39). The pathway leading to activation of ERK can be triggered by a variety of stimuli, including osmotic stress, and has been well elucidated in growth factor-stimulated cells: ERK is directly activated by mitogen-activated protein kinase kinase (MAPK kinase or MEK), and MEK is activated by Raf-1 kinase; Raf-1 is recruited to the membrane, where it is activated by Ras (25). Activation of ERK by hyperosmotic stress is reported to be mediated by Raf/MEK (33). However, it appears that Ras does not function as an activator of Raf in this case, because blocking Ras activation with its negative mutant, Ras-Asn17, did not affect ERK activation in response to hyperosmotic stress (7).
In addition to Ras, another recognized upstream activator of Raf is
protein kinase C (PKC) (5, 6, 21). PKC is a serine/threonine kinase,
and PKC isoforms have been divided into three categories on the basis
of their structure and biochemical properties: conventional PKC (cPKC),
including PKC, PKC
I,
PKC
II, and PKC
; novel PKC (nPKC), including PKC
, PKC
, PKC
, and PKC
; and atypical PKC (aPKC), including PKC
and PKC
/
. cPKC and nPKC are modulated by
phorbol ester and are also activated by diacylglycerol (DAG), whereas
aPKC is activated by phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3].
Endogenous DAG is derived from phosphatidylinositol 4,5-bisphosphate
following hydrolyzation by phospholipase C (PLC). This process is
accompanied by production of
D-myo-inositol
1,4,5-trisphosphate [Ins(1,4,5)P3],
a second messenger for mobilization of
Ca2+ from intracellular
endoplasmic reticulum stores that elevates cytosolic free
Ca2+, which is, in turn,
responsible for cPKC activation (29), whereas PtdIns(3,4,5)P3
is generated through the receptor-mediated activation of
phosphatidylinositol-3 kinase (PI-3 kinase) (36). It has been shown
that PKC plays a central role in the activation of ERK by a wide array
of stimuli (1, 16, 41). Involvement of PKC in transducing hyperosmotic
signals leading to activation of the ERK pathway was also demonstrated
by use of PKC inhibitors in MDCK cells (33). However, it is unclear
whether hyperosmotic stress activates PKC and which PKC isoforms are
involved in the ERK activation in response to this stress.
In this study, we investigated the effect of hypertonicity on PKC
activation and assessed the roles of cPKC and nPKC in activation of
ERK1 and ERK2 (ERK1/2) by hyperosmotic NaCl in NIH/3T3 mouse fibroblasts. This cell line was used because both cPKC and nPKC isoforms (PKC, PKC
, and PKC
) are expressed in this cell type (26) and because these three members have been shown to activate a
Raf-1/ERK pathway in fibroblasts (5, 6, 21). Our results show that
hyperosmotic NaCl increases total PKC activity and induces association
of PKC
, PKC
, and PKC
with cell membranes. Furthermore, we
demonstrate that ERK1/2 activation by NaCl is dependent on PLC and
cPKC/nPKC-mediated signaling.
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MATERIALS AND METHODS |
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Materials.
12-O-Tetradecanoylphorbol 13-acetate
(TPA) and platelet-derived growth factor (PDGF) were from R&D Systems.
LY-294002 and U-73122 were purchased from Biomol Research Lab.
GF-109203X and rottlerin were purchased from Alexis Biochemicals. A PKC
assay kit was purchased from GIBCO, and the DAG assay kit was from
Amersham. Rabbit polyclonal anti-MAPK R2 (CT) and sheep polyclonal
phospho-MAPK (ERK) were from Upstate Biotechnology. Phospho-p44/42 MAPK
(Thr202/Tyr204)
monoclonal antibody was from New England Biolabs. Anti-rabbit PKC
(319SA), PKC
(3147SA), and PKC
(13198-0150) antibodies were
obtained from Life Technologies. NaCl was from Fisher Scientific.
Cell culture.
NIH/3T3 cells and NIH/3T3 cells overexpressing PKC, PKC
, and
PKC
were maintained in DMEM supplemented with 7% calf serum. Cells
at 70-80% confluence were growth arrested by incubation in 0.5%
calf serum-DMEM for 24 h before use. For hypertonic treatment, NaCl
stock solution was added to the medium at the appropriate concentration.
Transient transfection and immunofluorescence microscopy.
Transient transfection and immunofluorescence were carried out as
described previously (40). Briefly, NIH/3T3 cells were seeded on plates
with coverslips and transiently transfected by the calcium phosphate
precipitation method with 6 µg of expression vector for full-length
mouse PKC, PKC
, or PKC
. After starvation, cells were
stimulated with 0.6 M NaCl for 5 min and then fixed with 3% (wt/vol)
paraformaldehyde in PBS and permeabilized with 0.1% Triton X-100. The
coverslips with cells were incubated with respective anti-PKC rabbit
monoclonal antibodies, followed by visualization with anti-rabbit IgG
FITC-labeled antibodies. Mounted cells were observed under a
fluorescence microscope (Nikon, Optiphoto 2/EFD2). In our previous
report, we showed that the disappearance of dark nuclei is a diagnostic
parameter indicating PKC translocation from the cytoplasm to the
membrane (27).
Establishment of cell lines overexpressing PKC isoforms.
NIH/3T3 cells seeded on 10-cm dishes (2 × 105 cells) were transfected with 1 µg of pSV2-Neo and 10 µg of expression vector for PKC, PKC
,
or PKC
by the calcium phosphate coprecipitation method (19). After 2 days of culture in 7% calf serum-DMEM, cells were selected with G418
(Geneticin; GIBCO) at a concentration of 300 µg/ml. After 7 days of
culture in the selection medium, colony-forming cells were picked up
and subcultured. The expression level of PKC isozymes for each
subculture was tested by Western blotting, and cell lines
overexpressing PKC
, PKC
, or PKC
were used for further experiments.
In vitro kinase assay.
The total PKC activity was determined by a PKC assay system following
procedures provided by GIBCO BRL. Briefly, treated or nontreated cells
on 10-cm dishes were washed with ice-cold PBS and harvested in
extraction buffer (20 mM Tris, pH 7.5, 0.5 mM EDTA, 0.5 mM EGTA, 0.5%
Triton X-100, and 25 µg/ml each aprotinin and leupeptin). After
homogenization, the homogenate was incubated on ice for 30 min and
centrifuged for 5 min at 6000 g. The
supernatant was used for detection of PKC activity with a PKC-specific
substrate, the synthetic amino-terminal acetylated peptide
corresponding to amino acids 4-14 of myelin basic protein
[Ac-MBP(414)] (38). The PKC-specific activity (pmol/min)
per assay tube was obtained after subtraction of nonspecific activity
(pmol/min) per assay tube with PKC inhibitor peptide and normalized to
total protein.
Western blot analysis.
Lysates were prepared and subjected to electrophoresis in 10%
SDS-polyacrylamide gel. After transfer of protein to a polyacrylidene difluoride membrane, the membranes were blocked with 5% (wt/vol) nonfat dry milk in 1× PBS overnight at 4°C. PKC, PKC
,
and PKC
were detected with their respective antibodies. Total ERK1/2
was detected by a rabbit anti-rat ERK antibody (anti-MAPK R2), and phosphorylated ERK1/2 was detected by phosphospecific ERK1/2
antibodies. The antibody-antigen complexes were detected using
horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG and
visualized by a standard chemiluminescence method performed according
to the manufacturer's instructions.
Cell fractionation. Cell fractionation was performed as described previously (40). Briefly, cells were washed with ice-cold PBS and harvested in lysis buffer. The lysate was sonicated for 20 s and then centrifuged at 1,300 g for 10 min. Supernatant was collected and centrifuged at 100,000 g for 40 min, the cytosol fraction was harvested, and the pellet was solubilized in cold lysis buffer containing 1% Nonidet P-40 and then centrifuged at 10,000 g for 10 min. This supernatant was used as the membrane fraction. Equal amounts of protein (20 µg/lane) were resolved by SDS-PAGE for subsequent immunoblot analysis.
DAG assay.
sn-1,2-DAG was measured with a DAG
assay reagent system following the instructions provided by Amersham.
Briefly, treated cells (1 × 106) were washed once with
ice-cold PBS without Ca2+ and
Mg2+ and harvested in 1 ml of PBS.
After centrifugation for 5 min at 1,500 rpm and 4°C, the cell
pellet was resuspended in 200 ml of cold PBS. Lipids were extracted,
and sn-1,2-DAG was radiolabeled using
DAG kinase and
[-32P]ATP. The
labeled sn-1,2-DAG was separated by
TLC and counted by scintillation counter (Beckman, LS 3801) for 4 min.
The amount of sn-1,2-DAG was
calculated from the mean counts per minute for each of the triplicate tubes.
Statistical analysis. Data were analyzed by the paired two-tailed Student's t-test.
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RESULTS |
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Comparison of ERK1/2 activation by hyperosmotic stress and TPA in
NIH/3T3 Cells.
It has been reported that hyperosmotic stress induces ERK activation in
several cell lines, including MDCK, 3Y1, and PC-12 cells (20, 33). TPA
is a strong activator of cPKCs and nPKCs and also induces ERK1/2
activation in a number of cell systems. To understand the role of PKC
in mediating ERK1/2 activation by hyperosmotic stress in NIH/3T3 cells,
we compared the activation of ERK1/2 by hyperosmotic NaCl and TPA.
ERK1/2 activation was measured by Western blotting with a
phosphospecific ERK1/2 antibody. When cells were incubated with 0.6 M
NaCl, ERK1/2 phosphorylation was detectable at 5 min and maximal at 25 min (Fig.
1A).
However, TPA (100 ng/ml) stimulated a very rapid increase in ERK1/2
phosphorylation that reached a maximum level within 5 min (Fig.
1A). Notably, the total cellular
ERK1/2 level remained constant through all of these time courses, as
shown by Western blotting with an antibody for ERK1/2 that detects both
the phosphorylated and unphosphorylated forms of the kinases.
Densitometric analysis revealed that the extent of ERK1/2
phosphorylation 25 min after NaCl incubation was similar to that in
TPA-treated cells at this time point (~7-fold relative to control;
Fig. 1B). The ERK activity induced
by either NaCl or TPA was sustained for at least 60 min with only a
slight decrease (data not shown). These results indicate that both
hyperosmotic stress and TPA strongly activate ERK1/2 in NIH/3T3 cells
but that the kinetics of ERK1/2 activation by hyperosmotic stress are
slower than those of activation by TPA.
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ERK1/2 activation by hyperosmotic stress depends on cPKC and nPKC.
cPKC and nPKC, but not aPKC, are sensitive to phorbol ester. Chronic
treatment of cells with TPA can downregulate or deplete cPKC and nPKC
isoforms. In NIH/3T3 cells, PKC is abundantly expressed, and PKC
,
PKC
, and PKC
are also detectable (26, 40). Previously, we showed
that incubation of cells with TPA (200 ng/ml) for 24 h could deplete
all of PKC
and most of PKC
and PKC
without affecting PKC
(40). To investigate whether cPKC and nPKC isoforms are required for
ERK1/2 activation by hyperosmotic stress, we preincubated cells with
TPA for 24 h and then exposed them to hyperosmotic NaCl. As shown in
Fig. 2, the prolonged TPA treatment completely abolished the activation of ERK1/2 by readdition of TPA,
verifying that downregulation of PKC is effective. Under this
condition, the phosphorylation of ERK1/2 by 0.6 or 1.0 M NaCl was also
largely inhibited, suggesting that certain isozymes of the cPKC and
nPKC groups do mediate activation of ERK1/2 induced by hyperosmotic
stress.
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Induction of PKC activity by hyperosmotic stress.
To test whether hyperosmotic stress activates PKCs, we first measured
total PKC activity in vitro using Ac-MBP(414) as a substrate. As
shown in Fig. 4, incubation of cells with
0.6 M NaCl caused a 2.6-fold increase in PKC activity compared with untreated cells at 10 min. This indicates that hyperosmotic stress can
induce PKC activation.
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Production of DAG following hyperosmotic stress.
Because activation of cPKC and nPKC is a consequence of DAG production
following PLC activation in intact cells, we assessed the ability of
hyperosmotic NaCl to stimulate DAG production using TLC. Figure
6 shows that treatment of cells with
hyperosmotic NaCl led to an increase in DAG production in a
dose-dependent manner, with a 3.5-fold increase in DAG levels in the
cells incubated with 1 M NaCl. Similar results were seen in
sorbitol-treated cells (data not shown), suggesting that hyperosmotic
stress induces PLC activation.
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PLC inhibitor, but not PI-3 kinase inhibitor, blocks the activation
of ERK1/2 by hyperosmotic stress.
Because PKC, PKC
, and PKC
are major intermediates in the
ERK1/2 activation pathway and because hyperosmotic stress also stimulates an increase in DAG levels, we next examined whether the
activation of PLC underlies the hyperosmolality-induced activation of
ERK1/2. As shown in Fig. 7,
A and
B, when cells were treated with
U-73122, a potent inhibitor of both PLC-
and -
(31), ERK1/2
phosphorylation stimulated by hyperosmotic NaCl was severely inhibited,
whereas only a small inhibitory effect was seen in cells treated with
LY-294002, a specific inhibitor for PI-3 kinase. Similarly, U-73122 but
not LY-294002 partially inhibited PDGF-stimulated ERK1/2
phosphorylation. In contrast, there was no inhibition of TPA-induced
phosphorylation of ERK1/2 by either inhibitor. These results suggest
that hyperosmotic stress-induced ERK1/2 phosphorylation is critically
dependent on a PLC-initiating signal transduction pathway in NIH/3T3
cells.
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DISCUSSION |
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In this report, we provide evidence that members of the cPKC and nPKC
groups are responsible for a major portion of the hyperosmotic stress-stimulated signal transduction that leads to the activation of
ERK1/2. We also demonstrate that hyperosmotic stress induces an
increase in total PKC activity and translocation of PKC, PKC
, and
PKC
from the cytosol to the membrane after the stimulation of
NIH/3T3 cells with NaCl.
The mechanism for PKC activation by growth factors is well established in mammalian cells, and our findings show that a partly common mechanism is also operating in hyperosmotic stress-induced PKC activation. Stimulation of tyrosine kinase receptors activates PLC and PI-3 kinase, leading to an increase in DAG and PtdIns(3,4,5)P3 levels, which in turn mediate activation of PKC (10, 29). Here we show that treatment of cells with hyperosmotic NaCl results in an increase in DAG levels (Fig. 6). Furthermore, it has been previously reported that hyperosmotic NaCl increases release of Ins(1,4,5)P3 and that urea induces PLC phosphorylation (30, 33). These results suggest that hyperosmotic stress triggers PLC activation, resulting in release of DAG and Ins(1,4,5)P3 and, as a consequence, PKC activation. It is likely that PLC and cPKC/nPKC lie along the same pathway, which mediates the activation of ERK1/2 by NaCl, because NaCl-induced ERK1/2 phosphorylation can be completely inhibited by either blocking PLC or TPA downregulation.
How PLC is activated by osmotic stress is currently unknown. Because
the activation of PLC is a consequence of phosphorylation of tyrosine
kinase receptors or activation of G protein-coupled receptors, one
possibility is that osmotic stress induces the activation of these
receptors, which then lead to activation of PLC. This notion is
supported by two studies, one of which shows that hyperosmotic sorbital
stimulates the aggregation of epidermal growth factor receptors (30)
and the other of which shows that urea-induced Egr-1 expression that is
mediated by PLC/PKC can be inhibited by genistein, a potent tyrosine
kinase inhibitor (10). However, Terada et al. (33) showed that ERK
activation by NaCl is insensitive to genistein, which argues against
tyrosine growth factor receptors being involved in this process.
Another possibility is that a non-membrane receptor/sensor-mediated
mechanism may be involved in the activation of PLC/PKC. Recently, it
was reported that a decrease in cell volume induces tyrosine
phosphorylation of several proteins (12) and that the activation of
PKC can be triggered by dehydration caused by hyperosmotic stress in
an in vitro system (12). Thus it is possible that shrinkage of cells by
extracellular hyperosmolality exerted by a nonpermeable solute such as
NaCl may directly activate these signaling molecules. Further studies
are needed to examine these two possibilities.
Among ten PKC isoforms reported, members of the cPKC and nPKC groups
seems to play the most essential roles in the activation of ERK1/2 by
hyperosmotic NaCl. This is suggested by several results obtained in
this work, as follows. First, the downregulation of PKC by prolonged
TPA treatment inhibits most of the phosphorylation of ERK1/2 by 0.6 or
1.0 M NaCl (Fig. 2). Second, highly selective cPKC and nPKC inhibitors
abrogate ERK1/2 phosphorylation (Fig. 3). Third, treatment of cells
with NaCl increases total PKC activity and induces association of
PKC, PKC
, and PKC
with the membrane (Figs. 4 and 5). Using
immunostaining analysis, we also observed aPKC (PKC
and PKC
)
translocation following NaCl treatment (data not shown). However, these
isozymes may not play a major role in NaCl-stimulated ERK1/2
activation, because the activation of ERK1/2 was severely reduced when
cPKC and nPKC were downregulated, leaving aPKCs intact (Fig. 2),
whereas inhibition of PI-3 kinase, an upstream activator of aPKC, by
LY-294002 had only a limited effect on NaCl-induced ERK1/2
phosphorylation (Fig. 7). Although our data suggest that members of
cPKC and nPKC groups present in NIH/3T3 cells mediate NaCl-induced
ERK1/2 activation, the contribution of individual PKC isoforms to
activation of these kinases remains to be defined.
The mechanism of PKC in activating ERK is currently being studied.
Increasing evidence indicates that PKC directly activates Raf-1 and
that Raf-1 then activates ERK through MEK. Kolch (21) reported that
PKC phosphorylates Raf-1, both in vitro and in vivo. Cacace et al.
(5) demonstrated that overexpressing PKC
in R6 rat fibroblasts
resulted in a marked increase in Raf-1 and MEK activity, and Cai et al.
(6) also showed that PKC
was able to directly activate Raf-1 in
vitro. Recently, we reported that another nPKC member, PKC
,
activates MEK in a Raf-dependent manner (35). These results, together
with our finding that NaCl treatment increases total PKC activity and
induces PKC
, PKC
, and PKC
translocation, suggest that these
PKC isoforms may activate ERK1/2 through a Raf/MEK pathway in cells
exposed to hyperosmotic stress.
The physiological significance of the activation of the ERK pathway by
osmotic stress in mammalian cells is still not clear. However, in
yeast, evidence has been presented for a role of MAPK pathways in
regulation of the transcriptional activation of the glycerol synthetic
pathway in response to high-salt conditions (2). In MDCK cells,
PKC-mediated ERK1/2 activation seems not to be required for
transcriptional stimulation of two osmolyte transporter genes,
myo-inositol and betaine, as osmolyte
transporter mRNA accumulation is still stimulated by hypertonicity
after PKC depletion (23). Nevertheless, the possibility cannot be ruled out that the PKC-ERK pathway may mediate the expression of other osmoregulatory genes by inducing transcription factors such as Egr-1
and c-Fos (8, 9, 13, 18, 32). On the other hand, it has been shown that
Na+-myo-inositol
cotransporter and
Na+-K+-Cl
cotransporter contain several PKC phosphorylation sites (24, 37) and
that the activation of PKC results in an increase of their activities
(11, 15). Activation of
Na+-myo-inositol
cotransporter is required for uptake of inositol, an organic solute
that plays an important role in adaptive regulation of long-term
hyperosmolality, whereas activation of
Na+-K+-Cl
cotransporter increases intracellular
K+ and
Na+, which initiate signals for
the induction of genes responsible for organic osmolyte accumulation
(3, 4). The functional roles of PKC isoforms in regulation of these
cotransporters remains unexplored. The finding that PKC
, PKC
, and
PKC
are activated in response to hyperosmotic NaCl lays the
groundwork for further study of PKC-mediated signaling events involving
regulation of cotransport systems, as well as the hypertonic activation
of genes.
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ACKNOWLEDGEMENTS |
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This work was supported in part by research grants from the Ministry of Education, Science, Sports, and Culture of Japan, the Cell Science Research Foundation, and the Uehara Memorial Foundation.
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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.
Address for reprint requests and other correspondence: S. Ohno, Dept. of Molecular Biology, Yokohama City University School of Medicine, 3-9, Fuku-ura, Kanazawa-Ku, Yokohama 236, Japan (E-mail: ohnos{at}med.yokohama-cu.ac.jp).
Received 5 April 1999; accepted in final form 26 August 1999.
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