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xanthine oxidase as a rat Paneth cell zinc-binding protein
Biochimica et Biophysica Acta 1540 (2001) 43^49 www.bba-direct.com
Identi¢cation of xanthine dehydrogenase/xanthine oxidase as a rat Paneth cell zinc-binding protein Yukari Morita a , Mitsutaka Sawada a; *, Hiroshi Seno a , Shigeo Takaishi a , Hiroaki Fukuzawa a , Naoki Miyake a , Hiroshi Hiai b , Tsutomu Chiba a a
Division of Gastroenterology and Hepatology, Department of Internal Medicine, Graduate School of Medicine, Kyoto University, 54 Shogoin-kawara-cho, Sakyo-ku, Kyoto 606-8507, Japan b Department of Pathology, Graduate School of Medicine, Kyoto University, Kyoto, Japan Received 8 February 2001; received in revised form 15 May 2001; accepted 15 May 2001
Abstract Paneth cells are zinc-containing cells localized in small intestinal crypts, but their function has not been fully elucidated. Previously, we showed that an intravenous injection of diphenylthiocarbazone (dithizone), a zinc chelator, induced selective killing of Paneth cells, and purified a zinc-binding protein in Paneth cells. In the present study, we further characterized one of these proteins, named zinc-binding protein of Paneth cells (ZBPP)-1. Partial amino acid sequences of ZBPP-1 showed identity with rat xanthine dehydrogenase (XD)/xanthine oxidase (XO). Anti-rat XD antibody (Ab) recognized ZBPP-1, and conversely anti ZBPP-1 Ab recognized 85 kDa fragment of rat XD in Western blotting. Messenger RNA and protein levels of XD were consistent with our previous data on the fluctuation of Paneth cell population after dithizone injection. Thus, ZBPP-1 is an 85 kDa fragment of XD/XO in Paneth cells. XD/XO in Paneth cells may play important roles in intestinal function. ß 2001 Elsevier Science B.V. All rights reserved. Keywords: Small intestine; Paneth cell; Xanthine dehydrogenase; Xanthine oxidase; Zinc; Reactive oxygen species
1. Introduction Paneth cells are located at the base in small intestinal crypts, and although they are morphologically well characterized, their physiological function has
Abbreviations: Ab, antibody; Abs, antibodies; DAB, diaminobenzidine tetrahydrocholoride; dithizone, diphenylthiocarbazone; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PVDF, polyvinylidene di£uoride; ROS, reactive oxygen species; XD, xanthine dehydrogenase; XO, xanthine oxidase; ZBPP, zincbinding protein of Paneth cells * Corresponding author. Fax: +81-75-751-4303. E-mail address: [email protected] (M. Sawada).
not been de¢ned completely. Paneth cells contain a number of bioactive substances including lysozyme [1], secretory phospholipase A2 [1], cryptidins (K-defensins) [1], matrilysin [2], IgA [3], tumor necrosis factor-K [4] and epidermal growth factor [5]. These facts suggest that Paneth cells have important physiological roles in the intestinal mucosa, especially in the mucosal defense mechanism. Our previous studies and other reports have revealed a high content of zinc in Paneth cell granules [6^8]. Intravenous administration of dithizone, a metal chelator with a high a¤nity to zinc, induced precipitation of purple-red zinc dithizonate complexes in Paneth cell granules [6]. Moreover, intra-
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venous injection of dithizone to rats killed Paneth cells rapidly and selectively, followed by their regeneration within 72 h [6]. Comparison of the intestinal proteins with or without dithizone treatment using sodium dodecyl sulfate^polyacrylamide gel electrophoresis (SDS^PAGE) showed the loss of several proteins from rat small intestine after dithizone injection, suggesting that those are the zinc-binding proteins in Paneth cells. Using a metal chelating af¢nity column saturated with zinc, two of those proteins were puri¢ed and were identi¢ed being localized in Paneth cells. One protein, zinc-binding protein of Paneth cells (ZBPP)-1, was approximately 90 kDa in molecular mass, and the second, ZBPP-2, was approximately 40 kDa. Monoclonal antibodies (Abs) raised against these ZBPPs speci¢cally stained Paneth cell cytoplasmic granules [9^11]. In addition, the antibody (Ab) against ZBPP-1 also stained mononuclear cells which showed immunoreactivity against CD68 Ab in lamina propria of the small intestine [11]. All of these results suggest that ZBPPs play important physiological roles in small intestinal function. Here, by partial sequencing of its amino acids, we found that ZBPP-1 in Paneth cells is a fragment of xanthine dehydrogenase (XD)/xanthine oxidase (XO).
with 100 mg of dithizone per kg of body weight through the tail vein over a period of 3 min under pentobarbital anesthesia without fasting. Control rats were injected with the solvent alone [6]. 2.3. Puri¢cation of ZBPP-1
2. Materials and methods
ZBPP-1 was puri¢ed as the methods described previously [9]. In brief, after euthanasia, the rat small intestinal mucosa was scraped with a razor and homogenized in 0.01 M phosphate bu¡er (PB) (pH 7.2) containing 1% Triton-X and 1 mM phenylmethylsulfonyl £uoride by sonication. The homogenates were centrifuged at 1600Ug for 20 min at 4³C, then the supernatant was applied to a chelating Sepharose column (Pharmacia, Uppsala, Sweden) saturated with zinc ion and equilibrated with 0.05 M Tris^acetic acid containing 0.5 M NaCl (pH 8.0). After the unbound material had been washed away, zinc-binding proteins were eluted with 0.05 M Tris^acetic acid containing 0.5 M NaCl (pH 3.0) and subsequently with 0.05 M EDTA containing 1 M NaCl. The eluate was dialyzed against 0.01 M PB overnight at 4³C and subsequently applied to a hydroxyapatite column (Seikagakukogyo Co., Tokyo, Japan) equilibrated with 0.01 M PB. Column bound proteins were eluted by stepwise increase of the PB concentration; 0.01, 0.05, 0.1, and 0.5 M, respectively. All fractions were dialyzed against 0.01 M PB overnight. The fraction eluted with 0.5 M PB contained ZBPP-1 [9].
2.1. Animals
2.4. Amino acid sequence analysis
Seven to 15-week-old male SPF Wistar rats, obtained from Shizuoka Laboratory Animal Center (Shizuoka, Japan), were kept in a stainless steel cage and given commercial rat chow as well as deionized water ad libitum.
The eluate containing ZBPP-1 was electrophoresed on a 10% polyacrylamide gel. Amino acid sequence analysis was performed by the in-gel digestion method described by Rosenfeld et al. [12] In brief, the strip of gel containing ZBPP-1 was excised from the gel and digested with bu¡er containing lysylendopeptidase (Wakojunyakukogyo Co., Osaka, Japan) at 35³C overnight. The digested protein was subsequently subjected to reversed phase high-performance liquid chromatography (HPLC). Two of the fractions separated by reversed-phase HPLC were used for the amino acid sequencing (HPG1005A Protein Sequencing Systems; Hewlett Packard Co., Palo Alto, CA).
2.2. Dithizone administration Dithizone (Merck, Darmstadt, Germany), a zinc chelator, was dissolved at a concentration of 10 mg/ml in saturated lithium carbonate solution (pH 11.8) and maintained at 70³C for 20 min. After cooling down to room temperature, the solution was ¢ltered and used immediately. All rats were injected
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2.5. Western blot analysis ZBPP-1 was analyzed by the Western blotting using anti-ZBPP-1 monoclonal Ab [9,11], and anti-rat xanthine dehydrogenase (XD) polyclonal Ab against rat liver XD kindly provided by Dr. Takeshi Nishino [14]. The eluate containing ZBPP-1 and recombinant XD [14] (a gift from Dr. Takeshi Nishino) were applied to SDS^PAGE and then electrophoretically transferred to a polyvinylidene di£uoride (PVDF) membrane (Immobilon-P; Millipore, Milford, MA), and analyzed by Western blotting with goat antimouse IgM for ZBPP-1, goat anti-rabbit IgG for XD as secondary Abs, and biotin^avidin^horseradish peroxidase complex (Vectastain Elite kit; Vector Laboratories, Burlingame, CA) as described previously [8]. The peroxidase activities were detected with 3,3P-diaminobenzidine (DAB, Dojindo, Kumamoto, Japan). To investigate the expression of XD after the dithizone administration, equivalent amounts of protein (20 Wg) from rat ileal mucosa at each time point were subjected to 10% SDS^PAGE and electrotransferred to PVDF membrane. Western blot analysis was undertaken as described above. 2.6. Northern blot analysis Total RNA was isolated from rat ileum at 0, 6, 12, 24 and 72 h and 1 week after dithizone administration using Trizol Reagent (Gibco BRL, Rockville, MD). Messenger RNA (mRNA) was isolated using Poly A Tract mRNA Isolation Systems (Promega, Madison, WI) from the total RNA as described above. Six Wg of mRNA was electrophoresed on 1.25% formaldehyde agarose gel and transferred to a nitrocellulose membrane (Schleicher and Schuell, Keene, NH). To make probes, the cDNAs of XD were synthesized from total RNAs of normal rat ileum by reverse transcriptase polymerase chain reaction using Super Script II reverse transcriptase^ polymerase chain reaction (RT^PCR) kit (Gibco BRL). The following primers were used. XD primers: sense 5P-TCATACCTTCTTCCCTGGCTAC-3P and antisense 5P-AAGCAAACAAACCCTGGCACCT-3P de¢ning a 746 bp product. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers: sense 5P-TTAGCCCCCCTGGCCAAGG-3P
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and antisense 5P-CTTACTCCTTGGAGGCCATG3P de¢ning a 556 bp product. The DNA probes were radiolabeled with [K-32 P]dCTP using random primer labeling kit (Amersham Pharmacia Biotech, Buckinghamshire, UK). The membrane was hybridized with the radiolabeled probe overnight at 65³C and washed with a stringent bu¡er, 0.2Usodium chloride/sodium citrate containing 0.1% SDS, at 65³C. The mRNA was quanti¢ed with a Fujix BAS 2000 image analyzer (Fujix, Tokyo, Japan) and normalized to GAPDH mRNA levels. Four independent experiments were performed. 2.7. Immunohistochemical procedure For the immunohistochemical study, the tissue samples were extirpated from rat ileum 2 cm proximal to the ileocecal junction without fasting. The samples were snap-frozen, and 7-Wm cryostat sections were subjected to immunostaining using the streptavidin^peroxidase technique [9]. The bound peroxidase was developed with DAB, and sections were counterstained with Mayer's hematoxylin. Speci¢city was assessed by preblocking the primary Ab with excess amount of recombinant XD. 3. Results 3.1. Amino acid sequencing of ZBPP-1 Several zinc-binding proteins were puri¢ed from Paneth cells using a¤nity column chromatography [9]. Among them, ZBPP-1 was sequenced from samples obtained from two independent fractions separated by reverse phase HPLC as a ¢nal puri¢cation step. The ¢rst sample was a 6-amino-acid peptide, Arg-Leu-Glu-Pro-Phe-Lys: the second, a 10-aminoacid peptide, Met-Leu-Gly-Val-Pro-Asp-Asn-ArgIle-Val. A database search using the BLAST program showed that the ¢rst and the second amino acid sequences are identical with 778^787 and 1099^1104 residues of rat XD, respectively, the enzyme which catalyzes the oxidation of hypoxanthine and xanthine to uric acid with concomitant reduction of NAD to NADH. XD is converted to XO by modi¢cation of the protein molecule; XO is one of the
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Fig. 1. Western blotting of ZBPP-1 and rat recombinant XD with anti-rat XD polyclonal Ab and anti-ZBPP-1 monoclonal Ab. (A) The eluate from hydroxyapatite column (lane 1) and rat recombinant XD (lane 2) were immunoblotted with anti-rat XD polyclonal Ab. (B) The eluate from hydroxyapatite column (lane 3) and rat recombinant XD (lane 4) were immunoblotted with anti-ZBPP-1 monoclonal Ab. Anti-ZBPP-1 and anti-XD Abs recognized the same proteins.
intrinsic factors which produces oxygen-derived free radicals in the small intestine [16,17]. Its presence in Paneth cell has not been recognized previously. 3.2. Western blot analysis As shown in Fig. 1, anti-rat XD polyclonal Ab recognized not only 145 kDa recombinant rat XD but also its 85 and 125 kDa fragments, and the Ab also recognized the puri¢ed ZBPP-1 (85 kDa). In addition, the Ab detected both 145 and 125 kDa proteins puri¢ed by a¤nity column chromatography saturated with zinc. The sizes of these proteins correspond exactly to those of rat XD and its 125 kDa fragment, respectively. Anti-ZBPP-1 monoclonal Ab also recognized not only the 145 kDa recombinant rat XD but also its 85 and 125 kDa fragments. Fur-
thermore, similar to anti-XD polyclonal Ab, antiZBPP-1 monoclonal Ab detected 145 and 125 kDa proteins puri¢ed by zinc a¤nity column in addition to the puri¢ed ZBPP-1 (85 kDa). Preincubation of the XD or ZBPP-1 primary Abs with rat recombinant XD or puri¢ed ZBPP-1, respectively, eliminated reactivity with 85, 125 and 145 kDa proteins (data not shown). These results con¢rm that rat Paneth cell ZBPP-1 corresponds to the 85 kDa fragment of XD. Moreover, the data also suggest that the 145 and 125 kDa proteins puri¢ed by a¤nity column chromatography saturated with zinc are rat XD and its fragment, respectively. XD protein expression changes in the intestinal mucosa following dithizone treatment as shown by Western blotting using anti-rat XD polyclonal Ab (Fig. 2). At 6 h after dithizone injection, the level of XD protein decreased dramatically compared with that in control rats. From 12 h to 1 week after dithizone injection, expression returned to the basal level. These results are consistent with our previous histological observation that Paneth cells regenerate within 72 h [6]. 3.3. Northern blot analysis Accumulation of XD mRNA level appears to precede Paneth cell regeneration. We hybridized mRNA isolated from rat ileum at times after dithizone injection to a 32 P-labeled probe speci¢c for XD. XD mRNA level started to increase immediately after Paneth cell shedding and reached a peak level within 12 h after dithizone administration. The signi¢cantly higher XD mRNA levels were maintained during the process of regeneration (12 and 24 h after dithizone administration) (Fig. 3). Our previous study using anti-ZBPP-1 monoclonal Ab revealed that recovery of ZBPP-1 protein levels in Paneth cells was complete within 72 h after dithizone injection [9,11].
Fig. 2. The protein levels of XD after dithizone administration detected by Western blotting. Equivalent amounts of protein (20 Wg) from rat ileum at 0, 6, 12, 24, and 72 h and 1 week after dithizone administration were subjected to SDS^PAGE, followed by immunoblot analysis with anti-rat XD polyclonal Ab. The expression level of XD decreased at 6 h after dithizone treatment and then increased at 24 h.
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3.4. Immunohistochemical evidence of XD in Paneth cells We also studied the immunohistochemical localization of XD in rat ileum using anti-rat XD polyclonal Ab. As shown in Fig. 4, cytoplasmic granules of Paneth cells were speci¢cally stained with anti-rat XD polyclonal Ab in un¢xed frozen sections. No other positively staining cells were observed. We con¢rmed the speci¢city of immunostaining using the primary Ab, preincubating with excess amount of recombinant XD; no positive staining was observed. Positive staining of Paneth cells disappeared 6 h after dithizone treatment and reappeared faintly in a few crypts at 72 h (Fig. 4). Therefore, we conclude that XD is a constituent of Paneth cell secretory granules. 4. Discussion
Fig. 3. Messenger RNA levels of XD in the rat ileum at 0, 6, 12, 24, and 72 h and 1 week after dithizone administration. Northern blot analysis was performed with 32 P-labeled XD cDNA. Lower panel shows quantitation values of XD mRNA expression normalized to GAPDH mRNA expression. Means þ S.D. from four separate experiments are shown. The relative level of XD mRNA was increased signi¢cantly at 12 and 24 h after dithizone administration. *P 6 0.01, **P 6 0.001 vs. 0 h.
Paneth cell granules contain a variety of bioactive substances, which may be involved in cell growth, di¡erentiation or apoptosis [4,5]. Paneth cell granules also contain several proteins contributing to mucosal immunity [1,3,13]. Indeed, it was recently con¢rmed that the dose-dependent secretion of K-defensin or cryptdins occurs within minutes in response to bacteria [13]. In addition to these substances, Paneth cells are known to possess large quantities of zinc in their granules. Our previous study showed that intravenous injection of dithizone, a zinc chelator,
Fig. 4. Immunohistochemical staining of XD using the tissue samples from rat ileum at 0, 6, and 72 h and 1 week after dithizone administration. The samples were incubated with a rabbit anti-rat XD antibody (1:100). (A) 0 h; (B) 6 h; (C) 72 h; (D) 1 week after dithizone administration. (E) 0 h using anti XD antibody pre-incubated with an excess amount of rat XD. Scale bars = 20 Wm.
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resulted in a rapid and selective killing of Paneth cells in association with the disappearance of several zinc binding proteins [9,10]. In the present study, we have identi¢ed ZBPP-1 as a fragment of XD/XO by amino acid sequence. Partial amino acid sequences from the two fragments of the HPLC-puri¢ed ZBPP-1 were found to have 100% homology with a part of the rat XD sequence. Furthermore, Western blot analysis revealed that anti-rat XD polyclonal Ab recognized ZBPP-1 and a larger protein puri¢ed by zinc a¤nity column chromatography, the molecular mass of which corresponded to native 145 kDa XD, and conversely that anti-ZBPP1 monoclonal Ab reacted with the native 145 kDa XD and its 85 kDa fragment. It was reported that rat XD has a molecular mass of about 145 kDa, which is mainly cleaved into three fragments of 20, 40, and 85 kDa by the protease, trypsin [15]. Moreover, recombinant rat XD is easily degraded to a 125 kDa protein by losing the 20 kDa fragment from its N-terminus (T. Nishino, unpublished data). Although it remains unknown at present whether XD is cleaved into these fragments in vivo, the data strongly suggest that ZBPP-1 is the 85 kDa fragment of rat XD. We have reported that Paneth cells disappeared from small intestinal crypts 6 h after dithizone treatment and began to reappear at 12 h [9]. In agreement with this change, we found in the present study by Western blot analysis that the XD protein level decreased 6 h after dithizone administration and increased thereafter. These results are also consistent with our previous and present immunohistochemical observations on Paneth cell changes after dithizone injection using anti-ZBPP-1 monoclonal Ab [9] and anti-rat XD polyclonal Ab, respectively. All of these results also support our ¢nding that ZBPP-1 is a fragment of XD. XD is converted to XO by modi¢cation of the protein molecule. This conversion occurs reversibly by oxidation of sulfhydryl residues or irreversibly by proteolysis [17]. The functional localization of XD and XO has been investigated by others using enzyme-histochemical as well as biochemical methods [18,19]. These enzyme activities were observed in various organs including the liver, kidney and alimentary tract, and the small intestinal epithelium showed a high levels of enzyme activity [18,19]. Our previous study using polyclonal Ab and monoclonal Ab
against ZBPP-1 showed immunoreactivity exclusively in Paneth cells and in mononuclear cells having CD68 immunoreactivity in the lamina propria of the small intestine [11]. In the present study, polyclonal Ab against rat liver XD, which cross-reacted with XO in Western blotting (data not shown), also revealed positive staining only in Paneth cell cytoplasmic granules. In contrast to our results, there are reports of XD activity in goblet cells and in other intestinal epithelial cells, and the XO activity was localized predominantly to the villous epithelia [18,19]. We cannot fully explain this apparent discrepancy. Perhaps the expression level of XD in Paneth cells is higher than that in other epithelial cells, resulting in the apparent exclusive staining of XD in Paneth cells under the conditions of our study. Alternatively, XD in Paneth cell granules may be more accessible to antibodies compared to XD from other cell lineages. XD catalyzes the conversion of hypoxanthine to xanthine and xanthine to uric acid in the purine catabolic pathway. Accordingly, inherited XD de¢ciency causes classical xanthinuria [20]; however, XD appears to have other physiological role [21]. Indeed, the conversion of hypoxanthine to xanthine by XD is associated with a concomitant reduction of NAD to NADH [22]. In this regard, it may be important to note that XD is the precursor of XO, and XO prefers using molecular oxygen, O2 , as an oxidizing substrate and produces H2 O2 and c O3 2 , potent oxygen radicals [21]. It has been reported that reactive oxygen species (ROS), including H2 O2 and c 3 O2 , have critical roles in the pathophysiology of tissue damage during an in£ammatory response [23], and that XO is one of the major molecules involved in ROS synthesis in the small intestine [24]. Our monoclonal Ab against ZBPP-1 showed immunoreactivity not only in Paneth cells but also in macrophages in the lamina propria, suggesting that XD/ XO might have some role concerning a respiratory burst in the macrophage [25]. In addition, it may be emphasized that ROS induce apoptosis through redox regulation [26,27]. Thus, in addition to the mucosal immunity XD or XO in Paneth cells may participate in the regulation of the turnover of epithelial cells in the small intestine by inducing apoptosis. On the other hand, our previous study using BrdU showed that dithizone administration induced a
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marked increase in the number of mitotic cells [28], suggesting that certain factors are released from Paneth cells to promote epithelial cell proliferation. Thus, it remains to be determined whether XD/XO in Paneth cells triggers a renewal of epithelial cells only by inducing apoptosis or also by producing certain factors which promote epithelial cell proliferation. To clarify the exact functional role of XD/XO in Paneth cells, we need to further investigate the expression of XD/XO in Paneth cells in various host conditions such as in£ammatory bowel disease. In conclusion, we report here that ZBPP-1 in Paneth cells is the 85 kDa fragment of XD/XO. This result provides a novel clue for understanding the role of Paneth cells not only in mucosal immunity but also in the regeneration of small intestinal epithelial cells. Acknowledgements This work was supported by a Grant-in-Aid for Scienti¢c Research from the Ministry of Culture and Science of Japan (11470130, 11877090 and 11670496) and a Grant-in-Aid for Research for the Future Program from the Japan Society for the Promotion of Science (JSPS-RFTF 97100201). We are grateful to Dr. Takeshi Nishino for his valuable suggestion and discussion; to Dr. Andre J. Ouellette for his critical reading of the manuscript; and to Mr. Haruyasu Koda for his technical assistance with histochemistry.
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[4] S. Keshav, L. Lawson, L.P. Chung, M. Stein, V.H. Perry, S. Gordon, J. Exp. Med. 71 (1990) 327^332. [5] S.S. Poulsen, E. Nexo, P.S. Olsen, J. Hess, P. Kirkegaard, Histochemistry 85 (1986) 389^394. [6] M. Sawada, K. Takahashi, S. Sawada, O. Midorikawa, Int. J. Exp. Pathol. 72 (1991) 407^421. [7] B. Stampel, Acta Histochem. (Jena) 8 (1959) 406^447. [8] D. Dinsdale, J. Histochem. Cytochem. 32 (1984) 139^145. [9] M. Sawada, M. Nishikawa, T. Adachi, O. Midorikawa, H. Hiai, Lab. Invest. 68 (1993) 338^344. [10] N. Miyake, M. Sawada, H. Hiai, Acta Histochem. Cytochem. 27 (1994) 357^363. [11] M. Sawada, Y. Horiguchi, P. Abujiang, N. Miyake, Y. Kitamura, O. Midoricawa, H. Hiai, J. Histochem. Cytochem. 42 (1994) 467^472. [12] J. Rosenfeld, J. Capdevielle, J.C. Guillemot, P. Ferrara, Anal. Biochem. 203 (1992) 173^179. [13] T. Ayabe, D.P. Satchell, C.L. Wilson, W.C. Park, M.E. Selsted, A.J. Ouellette, Nat. Immunol. 1 (2000) 113^118. [14] T. Ikegami, T. Nishino, Arch. Biochem. Biophys. 247 (1986) 254^260. [15] Y. Amaya, K. Yamazaki, M. Sato, K. Noda, T. Nishino, T. Nishino, J. Biol. Chem. 265 (1990) 14170^14175. [16] J.M. McCord, I. Fridovich, J. Biol. Chem. 243 (1968) 5753^ 5760. [17] T. Nishino, J. Biochem. (Tokyo) 116 (1994) 1^6. [18] A. Kooij, K.S. Bosch, W.M. Frederiks, C.J. Van-Noorden, Virchows Arch. B Cell. Pathol. Mol. Pathol. 62 (1992) 143^ 150. [19] Y. Moriwaki, T. Yamamoto, K. Yamaguchi, S. Takahashi, K. Higashino, Histochem. Cell Biol. 105 (1996) 71^79. [20] K. Ichida, N. Kamatani, T. Nishino, M. Saji, T. Hosoya, Adv. Exp. Med. Biol. 431 (1998) 327^330. [21] R.J. Korthuis, D.N. Granger, Clin. Cardiol. 16 (1993) 119^ 126. [22] F. Stirpe, E. Della-Corte, J. Biol. Chem. 244 (1969) 3855^ 3863. [23] M. Olyaee, S. Sontag, W. Salman, T. Schnell, S. Mobarhan, D. Eiznhamer, S. Keshavarzian, Gut 37 (1995) 168^173. [24] A. van-der-Vliet, T.J. Tuinstra, A. Bast, Biochem. Pharmacol. 38 (1989) 2807^2818. [25] S. Takao, E.H. Smith, D. Wang, C.K. Chan, G.B. Bulkley, A.S. Klein, Am. J. Physiol. 271 (1996) C1278^1284. [26] T.M. Johnson, Z.X. Yu, V.J. Ferrans, R.A. Lowenstein, T. Finkel, Proc. Natl. Acad. Sci. USA 93 (1996) 11848^11852. [27] M.D. Jacobson, M.C. Ra¡, Nature 374 (1995) 814^816. [28] N. Miyake, M. Sawada, H. Hiai, Acta Histochem. Cytochem. 28 (1995) 549^553.
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Identi¢cation of xanthine dehydrogenase/xanthine oxidase as a rat Paneth cell zinc-binding protein Yukari Morita a , Mitsutaka Sawada a; *, Hiroshi Seno a , Shigeo Takaishi a , Hiroaki Fukuzawa a , Naoki Miyake a , Hiroshi Hiai b , Tsutomu Chiba a a
Division of Gastroenterology and Hepatology, Department of Internal Medicine, Graduate School of Medicine, Kyoto University, 54 Shogoin-kawara-cho, Sakyo-ku, Kyoto 606-8507, Japan b Department of Pathology, Graduate School of Medicine, Kyoto University, Kyoto, Japan Received 8 February 2001; received in revised form 15 May 2001; accepted 15 May 2001
Abstract Paneth cells are zinc-containing cells localized in small intestinal crypts, but their function has not been fully elucidated. Previously, we showed that an intravenous injection of diphenylthiocarbazone (dithizone), a zinc chelator, induced selective killing of Paneth cells, and purified a zinc-binding protein in Paneth cells. In the present study, we further characterized one of these proteins, named zinc-binding protein of Paneth cells (ZBPP)-1. Partial amino acid sequences of ZBPP-1 showed identity with rat xanthine dehydrogenase (XD)/xanthine oxidase (XO). Anti-rat XD antibody (Ab) recognized ZBPP-1, and conversely anti ZBPP-1 Ab recognized 85 kDa fragment of rat XD in Western blotting. Messenger RNA and protein levels of XD were consistent with our previous data on the fluctuation of Paneth cell population after dithizone injection. Thus, ZBPP-1 is an 85 kDa fragment of XD/XO in Paneth cells. XD/XO in Paneth cells may play important roles in intestinal function. ß 2001 Elsevier Science B.V. All rights reserved. Keywords: Small intestine; Paneth cell; Xanthine dehydrogenase; Xanthine oxidase; Zinc; Reactive oxygen species
1. Introduction Paneth cells are located at the base in small intestinal crypts, and although they are morphologically well characterized, their physiological function has
Abbreviations: Ab, antibody; Abs, antibodies; DAB, diaminobenzidine tetrahydrocholoride; dithizone, diphenylthiocarbazone; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PVDF, polyvinylidene di£uoride; ROS, reactive oxygen species; XD, xanthine dehydrogenase; XO, xanthine oxidase; ZBPP, zincbinding protein of Paneth cells * Corresponding author. Fax: +81-75-751-4303. E-mail address: [email protected] (M. Sawada).
not been de¢ned completely. Paneth cells contain a number of bioactive substances including lysozyme [1], secretory phospholipase A2 [1], cryptidins (K-defensins) [1], matrilysin [2], IgA [3], tumor necrosis factor-K [4] and epidermal growth factor [5]. These facts suggest that Paneth cells have important physiological roles in the intestinal mucosa, especially in the mucosal defense mechanism. Our previous studies and other reports have revealed a high content of zinc in Paneth cell granules [6^8]. Intravenous administration of dithizone, a metal chelator with a high a¤nity to zinc, induced precipitation of purple-red zinc dithizonate complexes in Paneth cell granules [6]. Moreover, intra-
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venous injection of dithizone to rats killed Paneth cells rapidly and selectively, followed by their regeneration within 72 h [6]. Comparison of the intestinal proteins with or without dithizone treatment using sodium dodecyl sulfate^polyacrylamide gel electrophoresis (SDS^PAGE) showed the loss of several proteins from rat small intestine after dithizone injection, suggesting that those are the zinc-binding proteins in Paneth cells. Using a metal chelating af¢nity column saturated with zinc, two of those proteins were puri¢ed and were identi¢ed being localized in Paneth cells. One protein, zinc-binding protein of Paneth cells (ZBPP)-1, was approximately 90 kDa in molecular mass, and the second, ZBPP-2, was approximately 40 kDa. Monoclonal antibodies (Abs) raised against these ZBPPs speci¢cally stained Paneth cell cytoplasmic granules [9^11]. In addition, the antibody (Ab) against ZBPP-1 also stained mononuclear cells which showed immunoreactivity against CD68 Ab in lamina propria of the small intestine [11]. All of these results suggest that ZBPPs play important physiological roles in small intestinal function. Here, by partial sequencing of its amino acids, we found that ZBPP-1 in Paneth cells is a fragment of xanthine dehydrogenase (XD)/xanthine oxidase (XO).
with 100 mg of dithizone per kg of body weight through the tail vein over a period of 3 min under pentobarbital anesthesia without fasting. Control rats were injected with the solvent alone [6]. 2.3. Puri¢cation of ZBPP-1
2. Materials and methods
ZBPP-1 was puri¢ed as the methods described previously [9]. In brief, after euthanasia, the rat small intestinal mucosa was scraped with a razor and homogenized in 0.01 M phosphate bu¡er (PB) (pH 7.2) containing 1% Triton-X and 1 mM phenylmethylsulfonyl £uoride by sonication. The homogenates were centrifuged at 1600Ug for 20 min at 4³C, then the supernatant was applied to a chelating Sepharose column (Pharmacia, Uppsala, Sweden) saturated with zinc ion and equilibrated with 0.05 M Tris^acetic acid containing 0.5 M NaCl (pH 8.0). After the unbound material had been washed away, zinc-binding proteins were eluted with 0.05 M Tris^acetic acid containing 0.5 M NaCl (pH 3.0) and subsequently with 0.05 M EDTA containing 1 M NaCl. The eluate was dialyzed against 0.01 M PB overnight at 4³C and subsequently applied to a hydroxyapatite column (Seikagakukogyo Co., Tokyo, Japan) equilibrated with 0.01 M PB. Column bound proteins were eluted by stepwise increase of the PB concentration; 0.01, 0.05, 0.1, and 0.5 M, respectively. All fractions were dialyzed against 0.01 M PB overnight. The fraction eluted with 0.5 M PB contained ZBPP-1 [9].
2.1. Animals
2.4. Amino acid sequence analysis
Seven to 15-week-old male SPF Wistar rats, obtained from Shizuoka Laboratory Animal Center (Shizuoka, Japan), were kept in a stainless steel cage and given commercial rat chow as well as deionized water ad libitum.
The eluate containing ZBPP-1 was electrophoresed on a 10% polyacrylamide gel. Amino acid sequence analysis was performed by the in-gel digestion method described by Rosenfeld et al. [12] In brief, the strip of gel containing ZBPP-1 was excised from the gel and digested with bu¡er containing lysylendopeptidase (Wakojunyakukogyo Co., Osaka, Japan) at 35³C overnight. The digested protein was subsequently subjected to reversed phase high-performance liquid chromatography (HPLC). Two of the fractions separated by reversed-phase HPLC were used for the amino acid sequencing (HPG1005A Protein Sequencing Systems; Hewlett Packard Co., Palo Alto, CA).
2.2. Dithizone administration Dithizone (Merck, Darmstadt, Germany), a zinc chelator, was dissolved at a concentration of 10 mg/ml in saturated lithium carbonate solution (pH 11.8) and maintained at 70³C for 20 min. After cooling down to room temperature, the solution was ¢ltered and used immediately. All rats were injected
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2.5. Western blot analysis ZBPP-1 was analyzed by the Western blotting using anti-ZBPP-1 monoclonal Ab [9,11], and anti-rat xanthine dehydrogenase (XD) polyclonal Ab against rat liver XD kindly provided by Dr. Takeshi Nishino [14]. The eluate containing ZBPP-1 and recombinant XD [14] (a gift from Dr. Takeshi Nishino) were applied to SDS^PAGE and then electrophoretically transferred to a polyvinylidene di£uoride (PVDF) membrane (Immobilon-P; Millipore, Milford, MA), and analyzed by Western blotting with goat antimouse IgM for ZBPP-1, goat anti-rabbit IgG for XD as secondary Abs, and biotin^avidin^horseradish peroxidase complex (Vectastain Elite kit; Vector Laboratories, Burlingame, CA) as described previously [8]. The peroxidase activities were detected with 3,3P-diaminobenzidine (DAB, Dojindo, Kumamoto, Japan). To investigate the expression of XD after the dithizone administration, equivalent amounts of protein (20 Wg) from rat ileal mucosa at each time point were subjected to 10% SDS^PAGE and electrotransferred to PVDF membrane. Western blot analysis was undertaken as described above. 2.6. Northern blot analysis Total RNA was isolated from rat ileum at 0, 6, 12, 24 and 72 h and 1 week after dithizone administration using Trizol Reagent (Gibco BRL, Rockville, MD). Messenger RNA (mRNA) was isolated using Poly A Tract mRNA Isolation Systems (Promega, Madison, WI) from the total RNA as described above. Six Wg of mRNA was electrophoresed on 1.25% formaldehyde agarose gel and transferred to a nitrocellulose membrane (Schleicher and Schuell, Keene, NH). To make probes, the cDNAs of XD were synthesized from total RNAs of normal rat ileum by reverse transcriptase polymerase chain reaction using Super Script II reverse transcriptase^ polymerase chain reaction (RT^PCR) kit (Gibco BRL). The following primers were used. XD primers: sense 5P-TCATACCTTCTTCCCTGGCTAC-3P and antisense 5P-AAGCAAACAAACCCTGGCACCT-3P de¢ning a 746 bp product. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers: sense 5P-TTAGCCCCCCTGGCCAAGG-3P
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and antisense 5P-CTTACTCCTTGGAGGCCATG3P de¢ning a 556 bp product. The DNA probes were radiolabeled with [K-32 P]dCTP using random primer labeling kit (Amersham Pharmacia Biotech, Buckinghamshire, UK). The membrane was hybridized with the radiolabeled probe overnight at 65³C and washed with a stringent bu¡er, 0.2Usodium chloride/sodium citrate containing 0.1% SDS, at 65³C. The mRNA was quanti¢ed with a Fujix BAS 2000 image analyzer (Fujix, Tokyo, Japan) and normalized to GAPDH mRNA levels. Four independent experiments were performed. 2.7. Immunohistochemical procedure For the immunohistochemical study, the tissue samples were extirpated from rat ileum 2 cm proximal to the ileocecal junction without fasting. The samples were snap-frozen, and 7-Wm cryostat sections were subjected to immunostaining using the streptavidin^peroxidase technique [9]. The bound peroxidase was developed with DAB, and sections were counterstained with Mayer's hematoxylin. Speci¢city was assessed by preblocking the primary Ab with excess amount of recombinant XD. 3. Results 3.1. Amino acid sequencing of ZBPP-1 Several zinc-binding proteins were puri¢ed from Paneth cells using a¤nity column chromatography [9]. Among them, ZBPP-1 was sequenced from samples obtained from two independent fractions separated by reverse phase HPLC as a ¢nal puri¢cation step. The ¢rst sample was a 6-amino-acid peptide, Arg-Leu-Glu-Pro-Phe-Lys: the second, a 10-aminoacid peptide, Met-Leu-Gly-Val-Pro-Asp-Asn-ArgIle-Val. A database search using the BLAST program showed that the ¢rst and the second amino acid sequences are identical with 778^787 and 1099^1104 residues of rat XD, respectively, the enzyme which catalyzes the oxidation of hypoxanthine and xanthine to uric acid with concomitant reduction of NAD to NADH. XD is converted to XO by modi¢cation of the protein molecule; XO is one of the
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Fig. 1. Western blotting of ZBPP-1 and rat recombinant XD with anti-rat XD polyclonal Ab and anti-ZBPP-1 monoclonal Ab. (A) The eluate from hydroxyapatite column (lane 1) and rat recombinant XD (lane 2) were immunoblotted with anti-rat XD polyclonal Ab. (B) The eluate from hydroxyapatite column (lane 3) and rat recombinant XD (lane 4) were immunoblotted with anti-ZBPP-1 monoclonal Ab. Anti-ZBPP-1 and anti-XD Abs recognized the same proteins.
intrinsic factors which produces oxygen-derived free radicals in the small intestine [16,17]. Its presence in Paneth cell has not been recognized previously. 3.2. Western blot analysis As shown in Fig. 1, anti-rat XD polyclonal Ab recognized not only 145 kDa recombinant rat XD but also its 85 and 125 kDa fragments, and the Ab also recognized the puri¢ed ZBPP-1 (85 kDa). In addition, the Ab detected both 145 and 125 kDa proteins puri¢ed by a¤nity column chromatography saturated with zinc. The sizes of these proteins correspond exactly to those of rat XD and its 125 kDa fragment, respectively. Anti-ZBPP-1 monoclonal Ab also recognized not only the 145 kDa recombinant rat XD but also its 85 and 125 kDa fragments. Fur-
thermore, similar to anti-XD polyclonal Ab, antiZBPP-1 monoclonal Ab detected 145 and 125 kDa proteins puri¢ed by zinc a¤nity column in addition to the puri¢ed ZBPP-1 (85 kDa). Preincubation of the XD or ZBPP-1 primary Abs with rat recombinant XD or puri¢ed ZBPP-1, respectively, eliminated reactivity with 85, 125 and 145 kDa proteins (data not shown). These results con¢rm that rat Paneth cell ZBPP-1 corresponds to the 85 kDa fragment of XD. Moreover, the data also suggest that the 145 and 125 kDa proteins puri¢ed by a¤nity column chromatography saturated with zinc are rat XD and its fragment, respectively. XD protein expression changes in the intestinal mucosa following dithizone treatment as shown by Western blotting using anti-rat XD polyclonal Ab (Fig. 2). At 6 h after dithizone injection, the level of XD protein decreased dramatically compared with that in control rats. From 12 h to 1 week after dithizone injection, expression returned to the basal level. These results are consistent with our previous histological observation that Paneth cells regenerate within 72 h [6]. 3.3. Northern blot analysis Accumulation of XD mRNA level appears to precede Paneth cell regeneration. We hybridized mRNA isolated from rat ileum at times after dithizone injection to a 32 P-labeled probe speci¢c for XD. XD mRNA level started to increase immediately after Paneth cell shedding and reached a peak level within 12 h after dithizone administration. The signi¢cantly higher XD mRNA levels were maintained during the process of regeneration (12 and 24 h after dithizone administration) (Fig. 3). Our previous study using anti-ZBPP-1 monoclonal Ab revealed that recovery of ZBPP-1 protein levels in Paneth cells was complete within 72 h after dithizone injection [9,11].
Fig. 2. The protein levels of XD after dithizone administration detected by Western blotting. Equivalent amounts of protein (20 Wg) from rat ileum at 0, 6, 12, 24, and 72 h and 1 week after dithizone administration were subjected to SDS^PAGE, followed by immunoblot analysis with anti-rat XD polyclonal Ab. The expression level of XD decreased at 6 h after dithizone treatment and then increased at 24 h.
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3.4. Immunohistochemical evidence of XD in Paneth cells We also studied the immunohistochemical localization of XD in rat ileum using anti-rat XD polyclonal Ab. As shown in Fig. 4, cytoplasmic granules of Paneth cells were speci¢cally stained with anti-rat XD polyclonal Ab in un¢xed frozen sections. No other positively staining cells were observed. We con¢rmed the speci¢city of immunostaining using the primary Ab, preincubating with excess amount of recombinant XD; no positive staining was observed. Positive staining of Paneth cells disappeared 6 h after dithizone treatment and reappeared faintly in a few crypts at 72 h (Fig. 4). Therefore, we conclude that XD is a constituent of Paneth cell secretory granules. 4. Discussion
Fig. 3. Messenger RNA levels of XD in the rat ileum at 0, 6, 12, 24, and 72 h and 1 week after dithizone administration. Northern blot analysis was performed with 32 P-labeled XD cDNA. Lower panel shows quantitation values of XD mRNA expression normalized to GAPDH mRNA expression. Means þ S.D. from four separate experiments are shown. The relative level of XD mRNA was increased signi¢cantly at 12 and 24 h after dithizone administration. *P 6 0.01, **P 6 0.001 vs. 0 h.
Paneth cell granules contain a variety of bioactive substances, which may be involved in cell growth, di¡erentiation or apoptosis [4,5]. Paneth cell granules also contain several proteins contributing to mucosal immunity [1,3,13]. Indeed, it was recently con¢rmed that the dose-dependent secretion of K-defensin or cryptdins occurs within minutes in response to bacteria [13]. In addition to these substances, Paneth cells are known to possess large quantities of zinc in their granules. Our previous study showed that intravenous injection of dithizone, a zinc chelator,
Fig. 4. Immunohistochemical staining of XD using the tissue samples from rat ileum at 0, 6, and 72 h and 1 week after dithizone administration. The samples were incubated with a rabbit anti-rat XD antibody (1:100). (A) 0 h; (B) 6 h; (C) 72 h; (D) 1 week after dithizone administration. (E) 0 h using anti XD antibody pre-incubated with an excess amount of rat XD. Scale bars = 20 Wm.
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resulted in a rapid and selective killing of Paneth cells in association with the disappearance of several zinc binding proteins [9,10]. In the present study, we have identi¢ed ZBPP-1 as a fragment of XD/XO by amino acid sequence. Partial amino acid sequences from the two fragments of the HPLC-puri¢ed ZBPP-1 were found to have 100% homology with a part of the rat XD sequence. Furthermore, Western blot analysis revealed that anti-rat XD polyclonal Ab recognized ZBPP-1 and a larger protein puri¢ed by zinc a¤nity column chromatography, the molecular mass of which corresponded to native 145 kDa XD, and conversely that anti-ZBPP1 monoclonal Ab reacted with the native 145 kDa XD and its 85 kDa fragment. It was reported that rat XD has a molecular mass of about 145 kDa, which is mainly cleaved into three fragments of 20, 40, and 85 kDa by the protease, trypsin [15]. Moreover, recombinant rat XD is easily degraded to a 125 kDa protein by losing the 20 kDa fragment from its N-terminus (T. Nishino, unpublished data). Although it remains unknown at present whether XD is cleaved into these fragments in vivo, the data strongly suggest that ZBPP-1 is the 85 kDa fragment of rat XD. We have reported that Paneth cells disappeared from small intestinal crypts 6 h after dithizone treatment and began to reappear at 12 h [9]. In agreement with this change, we found in the present study by Western blot analysis that the XD protein level decreased 6 h after dithizone administration and increased thereafter. These results are also consistent with our previous and present immunohistochemical observations on Paneth cell changes after dithizone injection using anti-ZBPP-1 monoclonal Ab [9] and anti-rat XD polyclonal Ab, respectively. All of these results also support our ¢nding that ZBPP-1 is a fragment of XD. XD is converted to XO by modi¢cation of the protein molecule. This conversion occurs reversibly by oxidation of sulfhydryl residues or irreversibly by proteolysis [17]. The functional localization of XD and XO has been investigated by others using enzyme-histochemical as well as biochemical methods [18,19]. These enzyme activities were observed in various organs including the liver, kidney and alimentary tract, and the small intestinal epithelium showed a high levels of enzyme activity [18,19]. Our previous study using polyclonal Ab and monoclonal Ab
against ZBPP-1 showed immunoreactivity exclusively in Paneth cells and in mononuclear cells having CD68 immunoreactivity in the lamina propria of the small intestine [11]. In the present study, polyclonal Ab against rat liver XD, which cross-reacted with XO in Western blotting (data not shown), also revealed positive staining only in Paneth cell cytoplasmic granules. In contrast to our results, there are reports of XD activity in goblet cells and in other intestinal epithelial cells, and the XO activity was localized predominantly to the villous epithelia [18,19]. We cannot fully explain this apparent discrepancy. Perhaps the expression level of XD in Paneth cells is higher than that in other epithelial cells, resulting in the apparent exclusive staining of XD in Paneth cells under the conditions of our study. Alternatively, XD in Paneth cell granules may be more accessible to antibodies compared to XD from other cell lineages. XD catalyzes the conversion of hypoxanthine to xanthine and xanthine to uric acid in the purine catabolic pathway. Accordingly, inherited XD de¢ciency causes classical xanthinuria [20]; however, XD appears to have other physiological role [21]. Indeed, the conversion of hypoxanthine to xanthine by XD is associated with a concomitant reduction of NAD to NADH [22]. In this regard, it may be important to note that XD is the precursor of XO, and XO prefers using molecular oxygen, O2 , as an oxidizing substrate and produces H2 O2 and c O3 2 , potent oxygen radicals [21]. It has been reported that reactive oxygen species (ROS), including H2 O2 and c 3 O2 , have critical roles in the pathophysiology of tissue damage during an in£ammatory response [23], and that XO is one of the major molecules involved in ROS synthesis in the small intestine [24]. Our monoclonal Ab against ZBPP-1 showed immunoreactivity not only in Paneth cells but also in macrophages in the lamina propria, suggesting that XD/ XO might have some role concerning a respiratory burst in the macrophage [25]. In addition, it may be emphasized that ROS induce apoptosis through redox regulation [26,27]. Thus, in addition to the mucosal immunity XD or XO in Paneth cells may participate in the regulation of the turnover of epithelial cells in the small intestine by inducing apoptosis. On the other hand, our previous study using BrdU showed that dithizone administration induced a
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marked increase in the number of mitotic cells [28], suggesting that certain factors are released from Paneth cells to promote epithelial cell proliferation. Thus, it remains to be determined whether XD/XO in Paneth cells triggers a renewal of epithelial cells only by inducing apoptosis or also by producing certain factors which promote epithelial cell proliferation. To clarify the exact functional role of XD/XO in Paneth cells, we need to further investigate the expression of XD/XO in Paneth cells in various host conditions such as in£ammatory bowel disease. In conclusion, we report here that ZBPP-1 in Paneth cells is the 85 kDa fragment of XD/XO. This result provides a novel clue for understanding the role of Paneth cells not only in mucosal immunity but also in the regeneration of small intestinal epithelial cells. Acknowledgements This work was supported by a Grant-in-Aid for Scienti¢c Research from the Ministry of Culture and Science of Japan (11470130, 11877090 and 11670496) and a Grant-in-Aid for Research for the Future Program from the Japan Society for the Promotion of Science (JSPS-RFTF 97100201). We are grateful to Dr. Takeshi Nishino for his valuable suggestion and discussion; to Dr. Andre J. Ouellette for his critical reading of the manuscript; and to Mr. Haruyasu Koda for his technical assistance with histochemistry.
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