1 Second Department of Pathology, 2 Second Department of Surgery, and 3 Department of Parasitology, Miyazaki Medical College, 5200 Kihara, Kiyotake, Miyazaki 889-1692; and 4 Department of Life Science, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan
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ABSTRACT |
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Hepatocyte growth factor (HGF) activator inhibitor type 1 (HAI-1) and type 2 (HAI-2) are new Kunitz-type serine protease inhibitors that were recently purified and cloned from the human stomach cancer cell line MKN45 as specific inhibitors against HGF activator. Both proteins contain two Kunitz inhibitor domains and are expressed abundantly throughout the gastrointestinal tract, in addition to the placenta, pancreas, and kidney. In this study, to assess the possible roles of HAI-1 and HAI-2 in the intestinal mucosa, we examined the expression of HAI-1 and HAI-2 during regeneration of the intestinal mucosa. Immunohistochemical studies revealed that HAI-1 but not HAI-2 was detected more strongly in regenerative epithelium than in normal epithelium, although both proteins were detected throughout the human gastrointestinal tract. During the course of acetic acid-induced experimental colitis in an in vivo mouse model, HAI-1 but not HAI-2 was upregulated in the recovery phase, suggesting that HAI-1 but not HAI-2 is associated with the regeneration of damaged colonic mucosa. Upregulation of HAI-1 may serve to downregulate the proliferative response after initial activation of MET receptor by HGF/scatter factor after an injury.
hepatocyte growth factor; Kunitz-type proteinase inhibitor; mucosal injury; experimental colitis; immunohistochemistry
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INTRODUCTION |
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HEPATOCYTE GROWTH FACTOR (HGF)/scatter factor (SF) is a multifunctional polypeptide factor that is secreted by mesenchymal cells and functions as a mitogen, a morphogen, and/or a motogen for a variety of cells through MET receptor tyrosine kinase (2, 20). The HGF activator (HGFA) was identified as a serine proteinase that converts the inactive precursor of HGF to the active form (22, 35). HGFA is secreted mainly by the liver as an inactive zymogen, circulating in the blood in this form (22, 23, 34), and is activated by thrombin in injured tissue (23, 24, 33). The activity of HGFA is not inhibited by major plasma proteinase inhibitors, because HGFA is active in serum (22). Recently, as potent inhibitors of HGFA, two Kunitz-type serine proteinase inhibitors were purified from the conditioned medium of a human stomach cancer cell line, MKN45. These inhibitors were designated HGFA inhibitor type 1 (HAI-1) (32) and type 2 (HAI-2) (15), and both have two Kunitz-type serine proteinase inhibitor domains and a single putative transmembrane domain. Thus overall structure of the characteristic domains are similar between HAI-1 and HAI-2, except for the ligand-binding region of the low-density lipoprotein (LDL) receptor-like domain between the Kunitz domains of HAI-1 (32). A protein identical with HAI-2 was also reported by two other groups (18, 25).
Tissue distribution of HAI-1 is very similar to that of HAI-2 as assessed by RNA blot analysis, and both genes are expressed abundantly in the placenta, kidney, pancreas, and gastrointestinal tract (15, 32). Immunohistochemically, HAI-1 protein was detected in a simple columnar epithelium of ducts, tubules, and mucosal surface of various organs, including the gastrointestinal tract (13). HAI-1 proteins were localized on the lateral (or basolateral) cellular surface, and the expression of colonic epithelium was also confirmed by in situ hybridization (13). Expression of both HAI-1 and HAI-2 was conserved in colorectal adenocarcinomas, but their levels were decreased in poorly differentiated adenocarcinomas (12, 14). Because the MET receptor is frequently overexpressed in primary colon carcinomas (17), downregulation of both HAI-1 and HAI-2 may contribute to the malignant process through the increased activities of HGFA followed by the overactivation of HGF/SF.
In addition to the possible contribution of HGF/SF and its receptor MET, HGFA, and HAI-1 and -2 to colorectal adenocarcinomas, HGF/SF is upregulated in experimental gastric mucosal lesions (16, 31), promotes proliferation and migration (6), and accelerates the wound repair of cultured gastric mucosal cells (39). These results suggest that HGF/SF plays an important role in the repair of damaged gastrointestinal mucosa. In this study, therefore, to assess the possible roles of HAI-1 and HAI-2 in wound repair of damaged gastrointestinal mucosa, expression of HAI-1 and HAI-2 proteins in various inflammatory or ulcerative gastrointestinal diseases was examined immunohistochemically. We also cloned and sequenced the mouse homolog of HAI-1 (mHAI-1) by using a combination of RT-PCR and rapid amplification of cDNA ends (RACE) methods and examined the gene expression of both HAI-1 and HAI-2 during the course of mouse acetic acid-induced experimental colitis as an in vivo model. The results clearly showed the upregulation of HAI-1 but not HAI-2 in association with the regeneration of damaged mucosa.
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MATERIALS AND METHODS |
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Immunohistochemical analysis of HAI-1 in human gastrointestinal tissues. All human tissues were obtained from surgical or autopsied specimens of patients with various inflammatory or ulcerative gastrointestinal diseases. Formalin-fixed, paraffin-embedded sections were prepared according to a routine procedure. Deparaffinized and rehydrated serial 4-µm-thick sections were subjected to antigen retrieval by autoclaving for 5 min in 10 mM citrate buffer, pH 6.0. After treatment with 3% H2O2 in PBS for 10 min followed by washing in PBS twice, the sections were incubated with 3% BSA in PBS for 1 h at room temperature to block nonspecific binding. Subsequently, the sections were incubated with the primary mouse monoclonal antibody raised against human HAI (hHAI)-1 [1N7; 10 µg/ml in PBS containing 1% BSA (PBS/BSA)] or hHAI-2 (2N12; 10 µg/ml in PBS/BSA) at 4°C overnight (12, 13). Negative controls consisted of omission of the primary antibody, and the placenta, which expresses abundant HAI-1 and HAI-2 (15, 32), was used as positive control. For the adsorption test, the antibodies were pretreated with excess amounts of recombinant HAI-1 or HAI-2 protein. After being washed in PBS three times, the sections were incubated with Envision-labeled polymer reagents (DAKO, Carpinteria, CA) for 45 min at 37°C. After being washed in PBS three times, the sections were visualized with Ni,Co-3,3'-diaminobenzidine (ImmunoPure Metal Enhanced DAB Substrate Kit, Pierce, Rockford, IL) and counterstained with Mayer's hematoxylin.
Identification and cloning of mouse homolog of HAI-1 cDNA.
By screening the database of expressed sequence tags (ESTs) of the
National Center for Biological Information (NCBI) using the tBLASTn
algorithm, we identified two novel mouse cDNAs with Kunitz inhibitor
domains that are similar to hHAI-1 and hHAI-2 (11). On basis
of the results of a homology search using the BLASTn
algorithm, one cDNA seemed to be a mouse homolog of HAI-1 (mHAI-1),
although it is incomplete at 5', and the other cDNA seemed to be
a mouse homolog of HAI-2 (mHAI-2) (11). From the result of
the alignment of the ESTs, four primers, P1+, P1, P2
, and
P3
, were designed to obtain mHAI-1 cDNA containing the entire open reading frame by RT-PCR and 5'-RACE. One microgram of total cellular RNA from mouse kidney was reverse-transcribed by random hexamer [pd(N)6] and SuperScript II reverse transcriptase
(Life Technologies). The resulting cDNA was subjected to PCR for 35 cycles with the primers P1+ and P3
and HotStarTaq DNA polymerase (Qiagen, Valencia, CA). For 5'-RACE, Marathon-Ready mouse kidney cDNA (Clontech, Palo Alto, CA), which is an adaptor-ligated
double-strand cDNA, was used. Primers used for 5'-RACE were
P1
and AP1 (first-round PCR) and P2
and AP2 (second-round
PCR). The thermal cycle profile was 1 min at 94°C, 1 min
at 60°C, and 2 min at 72°C. Primer sequences were as follows:
P1+ (sense), 5'-ACTGTGCAGAGCTGCCAGAC-3'; P1
(antisense), 5'-AGGTGAATCGGGCACAGCGTTCACTGAAT-3'; P2
(antisense), 5'-TGGGATGTTCTCCTTGCAAAACCCAG-3'; P3
(antisense), 5'-AAGACTGGCAAGAGTCCCAG-3'; AP1,
5'-CCATCCTAATACGACTCACTATAGGGC-3'; AP2,
5'-ACTCACTATAGGG- CTCGAGCGGC-3'. The locations of RT-PCR
and 5'-RACE products are shown in Fig. 2A. After
electrophoresis, both 5'-RACE and RT-PCR products were cut from
agarose gel, purified with a QIAquick gel extraction kit (Qiagen), and
cloned into a TA cloning vector, pCR 2.1 (Invitrogen, San Diego, CA).
The nucleotide sequence was determined by ABI PRISM 310 Genetic
Analyzer using dye terminator cycle sequencing ready reaction kit
(Perkin Elmer, Foster City, CA). At least four individual clones each
from RT-PCR or 5'-RACE products were sequenced, and all
sequencing was done at least once in each direction to obtain maximum
accuracy for the sequence given.
Mice and RNA extraction. Eight- to ten-week-old male and late-phase pregnant female BALB/c mice maintained in specific pathogen-free conditions were obtained from Charles River Japan (Atsugi, Japan). Total cellular RNAs from various tissues of the mice were extracted by TRIzol (Life Technologies, Gaithersburg, MD), according to the manufacturer's protocol, immediately after death of these mice by cervical dislocation. Total cellular RNAs from mouse rectum at designated time points of experimental colitis, which was induced by intrarectal administration of 5% acetic acid under ether anesthesia, had been extracted previously (37). Briefly, the rectum was lavaged with 0.2 ml of saline for enema and then 0.1 ml of 5% acetic acid was administered into the rectum 3 cm proximal to the anus. Control mice were given 0.1 ml of saline in the same manner. Mice were killed 1, 3, and 5 days after administration, and the rectal tissues showing macroscopic erosion were used for histological examination and RNA extraction. Three mice were used at each designated time point, and the histological and RNA samples were prepared separately from each mouse.
RNA blot analysis.
Thirty micrograms each of total cellular RNA from various mouse tissues
and from mouse rectum at designated time points were denatured with
formamide, fractionated by electrophoresis on a 1% formaldehyde
agarose gel, and transferred onto Hybond-N nylon membrane
(Amersham, Little Chalfont, UK). Hybridization was performed under high-stringency conditions using 32P-labeled probes
made by the random-primed DNA labeling method as described previously
(9). The mHAI-1 probe used was a 5'-RACE product corresponding to
nucleotide position 1-1304, whereas the mHAI-2 probe used was an
RT-PCR product corresponding to nucleotide position 209-1082 (11).
Mouse HGF/SF, c-met, and HGFA probes were also made by PCR
using the following primer sets: mouse HGF/SF sense
5'-TTGGCCATGAATTTGACCTC-3' and antisense
5'-ACATCAGTCTCATTCACAGC-3' (40); mouse c-met sense
5'-GAATGTCGTCCTACACGGCC-3' and antisense 5'-CAGGGGCATTTCCATGTAGG-3' (40); and mouse HGFA sense
5'-CAAGGACTGTGGCACAGAGA-3' and antisense
5'-GATCTCTGTACCATTTCCCAGGAAGCA-3' (Ref. 8a; GenBank accession number AF099017). After hybridization, the
membrane was washed twice with 0.1× saline-sodium citrate
(SSC)-0.1% SDS at 65°C for 30 min and then exposed to Kodak XRP-5
films at 70°C for 7 days (mHAI-1), 5 days (mHAI-2), or
overnight [glyceraldehyde 3-phosphate dehydrogenase
(GAPDH)]. The blotted membrane was reprobed and be rehybridized
with a control human GAPDH probe (Clontech), which strongly
cross-hybridizes to mouse GAPDH mRNA. In the time course experiments,
the intensities of the mRNA signals of mHAI-1, mHAI-2, and GAPDH were
directly quantified by Bioimaging Analyzer, Fujix BAS 2000 system (Fuji
Photo Film, Tokyo, Japan) and the ratio of mHAI-1 and mHAI-2 to GAPDH
was calculated (37). The results are shown as means ± SD from three
mice at each designated time point and were analyzed using Student's
t-test. A P value of <0.05 was considered significant.
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RESULTS |
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Immunohistochemistry of HAI-1 and HAI-2 in human gastrointestinal
tissues.
Immunohistochemically, HAI-1 protein was diffusely detected in the
cellular surface of tall columnar epithelial cells, whereas HAI-2
protein showed granular intracytoplasmic staining, predominantly beneath the apical surface, of the epithelial cells, throughout normal
gastrointestinal tracts. Histiocytic mononuclear cells in the lamina
propria were also positively stained with HAI-2 in part, but other
stromal components such as fibroblasts, smooth muscle cells, and
endothelial cells were negative for both HAI-1 and HAI-2.
Interestingly, HAI-1 but not HAI-2 immunoreactivity was strongly
upregulated in the epithelial cells adjacent to the ulcer edge, which
are considered to be regenerative epithelial cells. Figure
1 shows an example of the results from a
specimen obtained from a colon surgically resected after polypectomy
because of malignancy. HAI-1 immunoreactivity was strongly upregulated in the ulcer edge (Fig. 1C) but not in normal mucosa far from the edge (Fig. 1B), whereas HAI-2 immunoreactivity was
unchanged in the corresponding regions (Fig. 1, E and
D, respectively).
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cDNA cloning, sequencing, and expression of mHAI-1.
To confirm the upregulation of HAI-1 at the transcriptional level
during the course of wound repair of damaged mucosa, we cloned and
sequenced mHAI-1 cDNA by using the NCBI database of ESTs. As a first
step toward obtaining the full-length mHAI-1 cDNA sequence, we used a
combination of the RT-PCR and 5'-RACE methods with total RNA of
BALB/c mouse kidney and the primers designed based on the alignment
result of ESTs (Fig.
2A). A total 2,228-bp
full-length mHAI-1 nucleotide sequence and its deduced amino acid
sequence were proposed by the combination of the nucleotide sequence of
RT-PCR and RACE products and those of ESTs corresponding to the
3' noncoding region (Fig. 2, A and
B). An ATG codon was found 155 nucleotide residues
downstream of the 5' end. A stop codon (numbered 1676), and the
following 563-bp noncoding region including a polyadenylation signal,
ATTAAA (numbered 2213-2218), were found at the 3' end. Thus
the peptide with 507 amino acids including two Kunitz-type serine
proteinase inhibitor domains [KD1 (Cys244 to
Cys294) and KD2 (Cys369 to
Cys419)], a putative LDL receptor-like domain (LR;
Cys309 to Cys347), and a putative transmembrane
domain [TM (Val444 to Phe466)] was
encoded by a single open reading frame. The first 29 amino acids
probably represent a signal peptide, based on the comparison with
hHAI-1 (32), whose cleavage sites of hHAI-1 (Ala35 and
Gln36) and mHAI-1 (Ala29 and Gly30)
satisfied the rules of von Heijne (38). However, hHAI-1 has an another
putative initiation ATG codon (Met1) six amino acids
upstream of the ATG (Met7) corresponding to the ATG
(Met1) of mHAI-1, so that hHAI-1 may have two distinctive
translation start sites. The nucleotide sequence of mHAI-1
corresponding to the ATG (Met1) of hHAI-1 is ACG but not
ATG. When the deduced amino acid sequence of mHAI-1 was compared with
that of hHAI-1, two Kunitz domains, an LR, a putative TM, and two
potential N-glycosylation sites were well conserved. The
identity of total amino acids and nucleotides between mHAI-1 and hHAI-1
was 80.7% and 82.6%, respectively. The amino acid identities of KD1,
LR, KD2, TM, and the COOH-terminal cytoplasmic region between mHAI-1
and hHAI-1 were 90.2%, 83.0%, 90.2%, 78.3%, and 87.8%,
respectively, suggesting that these well-conserved regions have
important roles in the function of HAI-1 protein. Indeed, the Kunitz
domains of mHAI-1 had less identity with those of other mouse
Kunitz-type serine proteinase inhibitors than the Kunitz domains of
hHAI-1 (25-29). Also, the LR of mHAI-1 had less identity with that
of other mouse LDL receptor-related molecules than the LR of hHAI-1 (3,
8, 26, 27). By RNA blot analysis, an mHAI-1 mRNA signal with an
approximate size of 2.2 kb was strongly detected in the colon, kidney,
and placenta and weakly in the stomach, duodenum, jejunum, and ileum
but was hardly detectable in the brain, heart, esophagus, liver,
testis, and ovary (Fig. 3). These results
were confirmed by two separate experiments using RNA preparations from
different mice. The expression pattern of mHAI-1 was basically
consistent with that of hHAI-1 (32) but apparently different from those
of hHAI-2, which is abundantly expressed in testis and ovary (15), and
other known Kunitz-type serine proteinase inhibitors (4, 10, 21, 36,
41).
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mHAI-1 and mHAI-2 expression during course of acetic acid-induced
experimental colitis.
Experimental colitis showing mucosal ulceration with marked
inflammatory infiltrate and hemorrhage was induced in the rectum 1 day
after administration of acetic acid and then gradually recovered to
normal by day 5 as shown in Fig. 4.
The mRNA signals and the expression ratio of mHAI-1 and mHAI-2 to GAPDH
at each designated time point are shown in Fig.
5. mHAI-1 expression was upregulated during
the recovery phase of colitis (days 3 and 5). The ratio of mHAI-1 to GAPDH expression was significantly increased at days 3 and 5 after treatment (P < 0.01 vs. day
0). On the other hand, mHAI-2 expression was not drastically
changed during the recovery phase of colitis (days 3 and
5), although it was downregulated once in the acute phase of
colitis (day 1). The ratio of mHAI-2 to GAPDH expression was
reduced to about one-third at day 1, but it was almost the same
between day 0 and the recovery phase (days 3 and
5). HGF/SF, c-met, and HGFA signals were consistently
detected in the rectal mucosa throughout the course of the experimental colitis, although the HGF/SF mRNA signal was upregulated on days 1-3 (Fig. 6). These results were
confirmed in three individual mice at each designated point.
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DISCUSSION |
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In this study, we demonstrate the upregulation of HAI-1 but not HAI-2 gene expression along with the regeneration of the intestinal mucosa by immunohistochemistry and analysis of acetic acid-induced experimental colitis in mice. Immunohistochemical studies using human colon tissue revealed an enhanced expression of HAI-1 but not HAI-2 in the regenerative epithelium compared with normal epithelium. Positive staining of HAI-1 was observed predominantly on the cellular surfaces (13), whereas that of HAI-2 was in the cytoplasm. Although the molecular structures of HAI-1 and HAI-2 are very similar, having two Kunitz domains and a transmembrane domain, the distinctive staining pattern of each HAI indicates the different subcellular localization of HAI-1 and HAI-2 and thus suggests the existence of a distinctive function of each HAI in the gastrointestinal tract. HAI-1 may be anchored on the plasma membrane by the transmembrane domain and its Kunitz domains would function in the extracellular environment, whereas HAI-2 may exist mainly in the cytoplasm anchoring in the intracellular compartments or may be secreted very quickly. In both HAIs, the first Kunitz domain is considered to be responsible for the inhibitory activity against HGFA (15, 29, 32) and the specific target proteinase for the second Kunitz domain of each HAI is currently uncertain. Therefore, the second Kunitz domain of each HAI may have distinctive important physiological target proteinases, and, consequently, HAI-1 and HAI-2 may have separate roles in the cellular biology of the gastrointestinal tract. In HAI-2, the inhibitory spectrum of the second Kunitz domain has been analyzed, showing strong inhibitory activities against trypsin, kallikrein, and plasmin, although this inhibitory spectrum was fairly similar to that of the first Kunitz domain (5).
As shown in Fig. 5, upregulation of HAI-1 but not HAI-2 was clearly represented during the course of acetic acid-induced experimental colitis in mice. mHAI-1 expression was upregulated during the recovery phase of the colitis (days 3 and 5). The ratio of mHAI-1 to GAPDH expression was significantly increased on days 3 and 5. Because most of the epithelial cells were damaged at day 1 (Fig. 4), the almost identical level of total mHAI-1 expression at days 0 and 1 may represent the increased mHAI-1 expression in the remaining epithelial cells. In parallel with the regeneration of the epithelial cells, total mHAI-1 expression was also upregulated significantly on days 3 and 5. Importantly, HGF/SF, c-met, and HGFA signals were consistently detected in the rectal mucosa throughout the course of the experimental colitis. The HGF/SF mRNA signal was upregulated on days 1-5, and this finding was basically consistent with previous reports (16, 31). The exact role of upregulated HAI-1 is uncertain at present. Because HGFA is produced in gastrointestinal tissue in addition to the liver (19) and an inactive form of HGFA is activated by thrombin in an injured tissue (23, 24, 33), various biological functions of HGF/SF, particularly epithelial cell proliferation and migration, might be initiated by the activated HGFA in this injured tissue. Excess HGF/SF and HGFA activated in the acute phase of injury may be inhibited by upregulated HAI-1 in the recovery phase, to avoid excess proliferation of the epithelial cells and to maintain homeostasis of gastrointestinal epithelium. This concept may also be supported by the fact that HAI-1 was downregulated in less differentiated adenocarcinomas of colon (14). Consistent suppression of inhibitory activities to HGFA and the following over-proliferation of epithelial cells may contribute to the malignant transformation of gastrointestinal epithelial cells. An alternative hypothesis is that HAI-1 may serve as a cell surface acceptor of activated HGFA, functioning on the cellular surface to localize the active HGFA on the cells that are going to enter the regenerative process. In fact, the binding between HAI-1 and HGFA seems to be reversible (unpublished observation), as observed in other Kunitz-type inhibitors (7).
On the other hand, mHAI-2 expression was not significantly altered during the recovery phase of colitis (days 3 and 5) compared with the preinjury state (day 0), although it was downregulated once in the acute phase of colitis (day 1). Reduction of mHAI-2 expression at day 1 may represent the number of damaged epithelial cells. Therefore, it is likely that mHAI-2 expression per epithelial cell is consistently unchanged during the course of experimental colitis. Recently, we identified and cloned mHAI-2 and its two novel splicing variants (11). Surprisingly, the spliced form lacking the entire KD1 coding region was the predominant transcript in mice but not in humans. Because the first domain of hHAI-2 is mainly responsible for the inhibitory activity against HGFA (29), these results may suggest that most of the mHAI-2 product expressed in mouse gastrointestinal tissue could not efficiently inhibit HGFA, although it is not obvious that KD1 of mHAI-2 protein is also responsible for the inhibitory activity against HGFA. The pathophysiological importance of this splicing is currently unknown. However, KD2 of HAI-2 might have additional important roles in gastrointestinal mucosa, because this region was consistently expressed in both mouse and human gastrointestinal epithelial cells, although HAI-2 is unlikely to contribute directly to the wound repair of damaged gastrointestinal mucosa.
In conclusion, we demonstrated the upregulation of HAI-1 but not HAI-2 gene expression during the regeneration of intestinal mucosa by immunohistochemistry and acetic acid-induced experimental colitis. HAI-1 may play an important role in the regeneration of damaged intestinal mucosa through the regulation of HGF/SF and HGFA, in addition to various other molecules associated with the regeneration such as growth factors other than HGF/SF, cytokines, and trefoil peptides (1, 28, 30). Further characterization of HAI-1 as well as HAI-2 in various pathophysiological conditions would be necessary to understand the in vivo function of these proteins in the gastrointestinal mucosa.
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ACKNOWLEDGEMENTS |
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The authors thank T. Miyamoto (preparation of figures), M. Kawamoto (nucleotide sequencing), and N. Iwakiri (preparation of histological sections) for excellent and skillful technical assistance.
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FOOTNOTES |
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This work was supported in part by Grant-in-Aid for Scientific Research (C) no. 11670221 from the Ministry of Education, Science, Sports, and Culture, Japan, and grants from the Kurozumi Medical Research Foundation and the Osaka Cancer Research Foundation.
The nucleotide sequence reported in this paper has been submitted to GenBank with accession number AF099018.
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: M. Koono, Miyazaki Medical College, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan (E-mail: koonom{at}post.miyazaki-med.ac.jp).
Received 7 September 1999; accepted in final form 8 November 1999.
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