Glycobiology Advance Access originally published online on October 19, 2007
Glycobiology 2008 18(1):20-27; doi:10.1093/glycob/cwm115
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Accelerated Proliferation and Abnormal Differentiation of Epidermal Keratinocytes in Endo-β-Galactosidase C Transgenic Mice
2 Division of Resources Life Science, United Graduate School of Agricultural Sciences; Tottori University, 4-101 Koyama-Minami, Tottori 680-8553, Japan
3 Animal Genome Research Unit, Division of Animal Science, National Institute of Agrobiological Sciences, 2 Ikenodai, Tsukuba, Ibaraki 305-0901, Japan
4 Department of Biological Science, Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu-cho, Matsue, Shimane 690-8504, Japan
5 Department of Health Science, Faculty of Psychological and Physical Sciences, Aichi Gakuin University; 12 Araike, Iwasaki-cho, Aichi 470-0195, Japan
1 To whom correspondence should be addressed: Tel: +81-852-32-6536; Fax: +81-852-32-6429; e-mail: tmatsu{at}life.shimane-u.ac.jp
Received on March 2, 2007; revised on September 27, 2007; accepted on October 5, 2007
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Abstract |
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Transgenic (TG) mice that have systemically expressed Endo-β-galactosidase C (EndoGalC) have rough and flaky skin. This skin phenotype is detectable around 5 days postnatal and becomes obscure by 2 weeks after birth. Their epidermis is thickened but the dermis and hair follicles are normal in structure. EndoGalC, which removes the terminal Gal
1-3Gal disaccharide (Gal epitope), was expressed in the epidermis of TG mice. GS-IB4 lectin staining showed that the Gal epitope did not exist in the epidermis in TG but existed in wild-type (WT) mice. In TG mice, N-acetylglucosamines were exposed by EndoGalC, which is detected using GS-II lectin. To understand the cause of the epidermal thickening and skin phenotype, we examined the proliferation and differentiation of kerationocytes. BrdU-pulse-labeling revealed that proliferating keratinocytes increased approximately three-fold in TG epidermis compared to WT one. In TG epidermis, the expression domain of cytokeratin 14 increased from 1–2 layers to 4–5 layers and co-expressed with cytokeratin 6 and 10 in the upper layers. The layers expressing involucrin and loricrin also increased but those expressing filaggrin and transglutaminase looked normal. The localization of E-cadherin was similar in both TG and WT mice. Although TG mice showed delayed development of the barrier function around 8 days postnatal, they acquired the function by 12 days after birth. These results suggest that the absence of the Gal epitope or the exposed N-acetylglucosamine terminal could play a critical role in the proliferation of basal keratinocytes and differentiation of them into the spinous cells in newborn mice. Key words: abnormal differentiation / endo-β-galactosidase C / epidermal hyperproliferation / keratinocyte / transgenic mice
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Introduction |
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Carbohydrates actively participate in developmental processes and also behave as potent antigens upon various immunological phenomena and diseases (Axford 1997
; Haltiwanger and Lowe 2004). However, precise roles of a large number of carbohydrates are still unknown. In this paper, we aimed at clarifying the action mechanism of Gal1-3Gal capping structure linked to N-acetylglucosamine terminal in glycoconjugates of the skin.
The Gal1-3Galβ1-4GlcNAc-R structure does not exist in human, apes, and old world monkeys and causes hyperacute rejection in xenotransplantation from nonprimate animals such as swine to human (Galili and Swanson 1991; Kobayashi and Cooper 1999). The hyperacute rejection is caused by natural antibodies directed against the Gal1-3Gal disaccharide (Gal epitope) expressed on the surface of donor's cells that are destroyed by antibody-mediated activation of complements (Auchincloss 1988; Kobayashi and Cooper 1999). The Gal epitopes are synthesized by sequential addition of two galactoses to a GlcNAc-R structure by β1,4galactosyltransferase (β4GalT) and 1,3galactosyltransferase (3GalT).
One of solutions of the hyperacute rejection is elimination or reduction of the terminal galactoses from nonprimate donors. For this purpose, mice lacking 3GalT were produced (Thall et al. 1995; Tearle et al. 1996). Although the mice showed disappearance of the reactivity for Griffonia simplicifolia isolectin-IB4 (GS-IB4) that specifically recognizes Gal epitope (reviewed by Ezzelarab and Cooper 2005), they exhibited no abnormality except for increased susceptibility to cataract (Tearle et al. 1996; Dahl et al. 2006). This could be partially explained by the existence of recently found another enzyme iGb3 synthase, which can synthesize the Gal epitope and compensate the loss of 3GalT (Milland et al. 2006).
As an alternative approach to diminish the Gal1-3Gal epitope, we produced transgenic (TG) mice expressing Endo-β-galactosidase C (EndoGalC) in the entire body. EndoGalC is an enzyme that specifically cleaves the linkage of Galβ1-4GlcNAc, resulting in the release of the Gal epitope (Fushuku et al. 1987; Ogawa et al. 2000). As shown in the accompanied paper, the TG mice showed the transient abnormal phenotype in the skin in infancy (Watanabe et al. submitted). This phenotype is also found in β4GalT1 knockout mice (Asano et al. 1997). The common phenotype is reasonable, since in both the TG and knockout mice, the exposed N-acetylglucosamine residue emerges and the Gal epitope is diminished. The detailed mechanism leading to the transient skin lesion remains to be clarified in β4GalT1 knockout mice. To elucidate the mechanism how the carbohydrate alteration in the skin affects the skin development, we performed the present investigation.
The skin consists of the epidermis, dermis, and hypodermis. The thickness of the hypodermis changes along with the hair cycle, but the epidermis and dermis keep constant thickness. The epidermis is usually classified into three nucleated layers, i.e., the basal (stratum basale), spinous (stratum spinosum), and granular layer (stratum granulosum), and one anucleated layer so-called the cornified layer (stratum corneum). The epidermal keratinocytes proliferate only in the basal layer under normal conditions, and the divided daughter cells stay in the basal layer as a stem cell or move up to the skin surface to be differentiated. The keratinocytes become gradually differentiated through the spinous, granular, and cornified layers, which can be distinguished by expression of specific proteins. In normal epidermis, cytokeratin 5 and 14 are only detected in the basal layer and cytokeratin 1 and 10 are expressed in the spinous and granular layers (Steinert 1993). Cytokeratin 6 and 16 do not exist in normal epidermis but appear in the spinous layer in hyperproliferating keratinocytes that are typically observed in healing wounds and skin diseases such as psoriasis (Baden et al. 1978; Mansbridge and Knapp 1987; Stoler et al. 1988; Iizuka et al. 2004; Koizumi et al. 2004). Involucrin is expressed in the upper part of the spinous layer and loricrin is observed in the granular layer (Watt 1983; Mehrel et al. 1990; Li et al. 2000). Profilaggrin is a large precursor containing multiple filaggrin repeats and is deposited in keratohyalin granules that are characteristics of the granular layer. During cornification, profilaggrins are dephosphorylated and fragmented into filaggrins which make intracellular bundles with cytokeratins (Ishida-Yamamoto et al. 2000). The cell envelopes are composed of involucrin, loricrin, elafin, and small proline-rich protein, which substitute for the cell membrane in the cornified cells. Transglutaminase increases the strength of the cell envelopes by coupling lysine and glutamine residues of involucrin and loricrin (Hitomi 2005).
The time required for differentiation of keratinocytes is usually constant but is vigorously shortened in hyperproliferating situations. In the human skin, the estimated turnover time of the epidermis is 40–56 days in normal skin and 6–8 days or shorter in psoriatic skin (Halprin 1972; Gelfant 1976). The turnover rate of epidermal keratinocytes must be correlated with their differentiation and thickness of the epidermis. Indeed, thickening of the epidermis is a typical feature of psoriasis.
Massive cell adhesion is one of the characteristics of the epidermis, which is mediated with specific cell surface molecules. The tight junction, one of the cell adhesion apparatuses, is conspicuous in the upper part of the granular layer and greatly takes part in the barrier function of the skin. Occludin and claudin are major components of tight junctions and therefore closely associated with cell adhesion (Gonzalez-Mariscal et al. 2003). Interestingly, the mice-overexpressed claudin 6 under the control of the involucrin promoter showed thickening of the epidermis and abnormal cornified envelopes in addition to reduction of the barrier function in the skin (Turksen and Troy 2002). Based on the above consideration, we examined morphology, cell proliferation, barrier function, and expression of various markers in the skin of EndoGalC TG mice.
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Results |
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Transgenic mice expressing EndoGalC have thickened epidermis
EndoGalC TG mice have rough skin with a lot of flakes of the cornified epidermal sheets, which is detectable around 5 days postnatal, but the lesion becomes unclear by 2 weeks after birth. Hematoxylin and eosin staining of the skin revealed that the epidermis of TG mice were five- to seven-fold thicker than that of WT mice 8 days after birth (Figure 1). In TG mice, the basal and suprabasal layers were more numerous (Figure 1A and B) and the stratum corneum had an increased number of tightly piled layers of cornified cells (Figure 1C and D), although the morphology and thickness of the dermis were similar to WT mice (Figure 1F and G). It was sometimes observed of TG mice that incompletely cornified keratinocytes appeared on the cornified layer (Figure 1E). However, the entire skin in TG mice was thin, because the hypodermis was less developed even in the hair-growing phase (anagen) (Figure 1F and G). The size and shape of epidermal keratinocytes were similar between TG and WT mice. In addition, no obvious difference was found in the organization of hair follicles and sebaceous glands between both mice.
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EndoGalC proteins were discontinuously expressed in the epidermis in TG mice 8 days after birth; the expressing areas looked like patches with various sizes (Figures 1H and 2A and B). FITC-labeled GS-IB4 that recognizes the
Gal epitope bound to the basal layer of epidermis in WT mice (Figure 2C) but hardly bound to that in TG mice (Figure 2A). Instead, the terminal N-acetylglucosamines were exposed in TG mice in the area where the terminal galactoses were decreased (Figure 2B), which were not observed in the epidermis of WT mice (Figure 2D). The expression of EndoGalC proteins in the epidermis diminished by 2 weeks after birth and was not detected in adult mice.
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Abnormalities were also found in tail and ventral skins of TG mice (Figure 3). The tail of TG mice often showed a segmented appearance, which had a swelling phenotype with some narrowed points along its length. The surface of the tail was rough and very flaky, which became marked from 5 days postnatal and persisted to around 2 weeks after birth. The ventral skin also had rough and flaky skin but the symptom was not severe than in the dorsal and tail skin. The tail and ventral skin had a thickened epidermis as well as the dorsal skin (Figure 3B and F). EndoGalC proteins were expressed at stronger levels and in more cells in the tail epidermis (Figure 3D) compared to the dorsal epidermis (Figures 1H and 2A and B). EndoGalC expression in the ventral epidermis was at an intermediate level between the tail and dorsal skin (Figure 3H).
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Expression of differentiation-associated proteins in EndoGalC TG mice
To identify the layer(s) of epidermis accounting for the thickened phenotype in TG mice, we performed immunohistochemistry with several differentiation markers for epidermal keratinocytes (Figure 4). Cytokeratin 14 is usually expressed in undifferentiated keratinocytes in the basal layer of the epidermis and hair follicles. Although WT epidermis generally possessed a single layer of basal cells (Figure 4E), cytokeratin 14 positive cells were broadly localized in four or five layers of the epidermis in TG mice (Figure 4F). The area expressing cytokeratin 10, a marker of differentiating keratinocytes, was also expanded in TG mice compared with WT mice (Figure 4G and H). In the epidermis of TG mice, cytokeratin 14 and 10 were colocalized in some layers. Moreover, TG epidermis also expressed cytokeratin 6 (Figure 4I and J) which was not found in a normal skin but appeared in hyperproliferating keratinocytes in the spinous layer in the case of wound healing or inflammation (Baden et al. 1978
; Mansbridge and Knapp 1987; Stoler et al. 1988; Iizuka et al. 2004; Koizumi et al. 2004). E-cadherin, an important cell adhesion molecule, was detected on the cell membrane of epidermal keratinocytes in both WT and TG mice (Figure 4K and L). The distribution pattern and the amount of E-cadeherin proteins were similar between WT and TG mice, although the expression area increased in TG mice because of increased number of keratinocytes. We also examined the expression of molecules associated with cornification such as involucrin, loricrin, filaggrin, and transgulutaminase. Involucrin and loricrin usually started to be synthesized in the spinous and granular layer, respectively. Their expression areas were increased in TG mice (Figure 4M–P). While the expression of filaggrin and transgulutaminase in TG mice were almost the same as that in WT mice and restricted to the cornified layer (Figure 4Q–T).
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Enhanced proliferation of epidermal cells
To understand the cause of thickening of TG epidermis, 5-bromo-2-deoxyuridine (BrdU) was administered before sampling the skin, and proliferating cells were detected with anti-BrdU antibody (Figure 5). Significantly a large number of proliferating cells were detected in the basal layer but not in the follicular tissues of TG mice compared to WT mice (Figure 5, Table I). The frequency of BrdU-positive cells in the bottom layer of the epidermis (basal layer) was 35.0% in TG mice and 10.3% in WT mice. In contrast to WT epidermis, BrdU-positive cells were also found in the cells above the bottom layer in the TG epidermis (Table I).
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Morphological difference of cornified cells
Cornified cells were isolated from the back skin 8 days postnatal and their morphology was observed under a light microscope (Figure 6). There was a variation in the sizes of the cells, which would reflect maturation levels of cornification. Cornified cells had polygonal and angular shapes in WT mice (Figure 6A and B) but round shapes in TG mice (Figure 6E and F) regardless of the cell size. Moreover, the cells, especially large and matured ones, appeared to be more tightly attached to each other in WT mice. To confirm the elimination of the
Gal epitopes by EndoGalC activities, the isolated cornified cells were reacted with FITC-labeled GS-IB4 lectin. The lectin bound on the surface of WT-derived cells (Figure 6D) but never to TG-derived ones (Figure 6H).
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Establishment of skin barrier function delayed in EndoGalC TG mice
Moisture contents on the surface of the dorsal skin were measured to assess the establishment of the barrier function in newborn mice. Siblings obtained by mating heterogenous TG mice were individually identified and subjected for the daily measurement of skin moisture contents from 7 to 14 days after birth. The siblings included homogenous TG, heterogenous TG, and WT mice. Moisture contents of WT mice decreased as age and settled at a constant low level by 10 days postnatal. TG mice also showed a decrease of moisture contents, but it delayed approximately 1 day in heterogenous TG and 2 days in homogenous TG compared to WT mice (Figure 7A). The delay seems to be relevant to the size of mice (Figure 7B). However, one WT mouse with a lightweight showed quick development of the barrier function almost equivalent to other WT mice (Figure 7).
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Discussion |
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Association of the carbohydrate altration in control of proliferation and differentiation of epidermal keratinocytes
EndoGalC TG mice had a rough and flaky skin and showed an accelerated proliferation and abnormal differentiation of epidermal keratinocytes. In TG mice, the epidermis became thick (Figure 1) and almost completely lost the
Gal epitopes from the surface of epidermal cells (Figures 2A and 6H). A rough skin and thickening of the epidermis are also reported in β4GalT1 knockout mice (Asano et al. 1997; Lu et al. 1997). The Gal epitope is expected to be decreased in β4GalT1 knockout mice, while the knockout mice and the TG mice share another carbohydrate profile, exposed N-acetylglucosamine residue as a result of incomplete synthesis or enzymatic digestion. Exposure of the N-acetylglucosamines was clearly shown in the basal layer of TG mice where the Gal epitopes were detected in WT mice (Figure 2B and C). This fact suggests that EndoGalC proteins expressed in the TG mice can efficiently cleave the linkage of Galβ1-4GlcNAc and expose the N-acetylglucosamine by releasing the Gal1-3Gal disaccharide. As the phenotypes of the β4GalT1 knockout mice and the EndoGalC TG mice were very similar, the skin abnormalities found in both the mice would be caused by diminishing the Gal epitopes or exposing the N-acetylglucosamines.
Cytokeratin 14 expressing keratinocytes were observed in multiple layers in the epidermis of TG mice and co-expressed cytokeratin 10 in its upper domain (Figure 4F and H). The number of BrdU-positive keratinocytes in the bottom layer of the epidermis in TG mice was approximately three times that in WT mice (Figure 5, Table I). Proliferating keratinocytes were also seen in the area above the bottom layer of the epidermis (Figure 3, Table I). These facts indicated that basal keratinocytes would vigorously proliferate and incompletely differentiate in TG mice since the growth of basal cells might be too rapid to differentiate into spinous cells appropriately. The keratinocytes expressing both cytokeratin 14 and 10 also began to synthesize cytokeratin 6 and were similar to immature spinous cells that had been found at wound edges (Mansbridge and Knapp 1987; Koizumi et al. 2004). In contrast, the number of BrdU-positive cells was not significantly different in the follicular epithelium between TG and WT mice (Figure 5). Therefore, the abnormal proliferation and differentiation of keratinocytes in TG mice were restricted to the interfollicular keratinocytes and might be caused by inflammatory cytokines relevant to wound healing.
Normal differentiation of keratinocytes in the cornified layer in TG mice
Though the area expressing involucrin or loricrin was expanded, the cornified layer containing filaggrin and transglutaminase looked normal (Figure 4). Localization of E-cadherin proteins was not different between WT and TG mice (Figure 4K and L), suggesting that cell adhesion might be normal in the epidermis of TG mice. In fact, the barrier function of the skin of TG mice was rapidly established after birth as well as that of WT mice, though it delayed 1 or 2 days (Figure 7). These results demonstrated that EndoGalC TG mice showed hyperplasia of epidermal keratinocytes but it was clearly different from psoriasis. In the typical psoriasis, loricin was not detected in the epidermis and involucrin was expressed in the lower portion of the spinous layer (Iizuka et al. 2004), which made a clear contrast with TG mice that expressed differentiation-associated molecules in proper domains though some expression areas became broader (Figure 4). Thus Gal epitope or masking of N-acetylglucosamine would play important roles in proliferation of basal keratinocytes and their differentiation in the spinous.
There were also some differences in the cornified layer of TG mice from WT one. The number of cornified cell layers was generally large in TG mice (Figure 1C). This might be caused by the accelerated growth of keratinocytes. As the certain duration is needed for cornification, hyperproliferation of keratinocytes should result in deposition of cornifing cells. In some TG mice, parakeratosis was found on the cornified layer (Figure 1E, arrows), which also indicates abnormal differentiation and deficient keratinization in TG mice.
Barrier function of the skin in TG mice is transiently deficient
The cornified cells of TG mice were slightly smaller in size and more rounded in shape, and their adhesiveness might be a little weaker than those of WT mice 8 days after birth (Figure 6). At that time, mice were in course of establishing their skin barrier function. TG mice showed delays of the development of the epidermal barrier 1 or 2 days compared to WT mice (Figure 7). The duration found that these delays well correspond with the critical period of neonatal death of TG mice, which was around 9 days after birth (Watanabe et al. submitted). However, the TG mice passed through the critical period looked normal and their barrier function of the skin was not substantially different from that of WT mice. These results indicate that cornification may be transiently deficient in TG mice from 7 to 11 days after birth. In claudin 6-overexpressing TG mice that were designed to express the transgene in the spinous layer, the epidermis and the entire skin become thick and the morphology of their cornified cells is similar to that of EndoGalC TG mice (Turksen and Troy 2002). As claudin 6 is relevant to the formation of tight junctions, it is suggested that Gal epitope or N-acetylglucosamine masking could also be associated with cell adhesion by controlling the functions of tight junctions.
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Materials and methods |
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Mice
Endo-β-galactosidase C (EndoGalC) TG mice were produced by injection of the β-actin promoter-driven endoGalC gene of Clostridiumu perfingens into fertilized eggs (Watanabe et al. submitted). TG-10 line of the TG mice was inbreeded and the offspring were genotyped by PCR. Homogenous TG mice could not be distinguished from heterogenous ones by genotyping, but may be distinguished by their extremely small size and severe phenotype. All experiments using mice were carried out along the guidelines for animal experimentation that had been instituted by Department of Experimental Animals Center for Integrated Research in Science of Shimane University.
Histological and immunohistological analysis
Eight-day-old mice were painlessly killed and their dorsal, ventral, and tail skins were isolated and fixed in 4% paraformaldehyde for 3 h at room temperature. Paraffin sections (7 µm thick) were stained with hematoxylin and eosin or subjected to immunohistochemistry. To retrieve antigenecity, sections were treated with citric buffer (10 mM, pH 6.0) for 40 min at 95°C. The treated paraffin sections and unfixed cryosections were incubated overnight at 4°C with primary antibody: 1/300 diluted anti-Endo-β-galactosidase rabbit polyclonal antibody (AE1), 1/100 diluted anticytokeratin 14 mouse monoclonal antibody (Abcam, Cambridge, UK), 1/100 diluted anticytokeratin 10 mouse monoclonal antibody (Millipore (Chemicon), Billerica, MA), 1/50 diluted anticytokeratin 6 mouse monoclonal antibody (Progen, Heidelberg, Germany), 1/500 diluted anti-E-cadherin mouse monoclonal antibody (R&D systems, Minneapolis, MN), 1/100 diluted anti-involucrin mouse monoclonal antibody (Sigma-Aldorich (Sigma), St. Louis, MO), 1/500 diluted antiloricrin rabbit polyclonal antibody (Abcam), 1/1000 diluted antifilaggrin rabbit polyclonal antibody (Covance, Berkeley, CA), or 1/50 diluted antitransglutaminase rabbit polyclonal antibody (GeneTex, San Antonio, TX). These specimens were reacted with 1/100 diluted Texas Red conjugated antimouse IgG antibody (EY Laboratories, San Mateo, CA) or 1/1000 diluted Alexa-Fluor 488-labeled antirabbit IgG antibody (Invitrogen (Molecular Probes), Carlsbad, CA) for 30 min at room temperature. The nuclei were stained with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) (Polyscience, Niles, IL). All fluorescent signals were captured with a chilled monochrome charge-coupled device (CCD) camera (Penguin 150CLM) (Pixera, Los Gatos, CA) and processed by pseude-color.
Detection of terminal galactose and N-acetylglucosamine with lectins
GS-IB4 was used to detect the presence of Gal1-3Gal disaccharide (Gal epitope) of the Gal1-3Galβ1-4GlcNAc-R structure. Cleavage of the Gal1-3Galβ1-4GlcNAc-R structure at Galβ1-4GlcNAc site by EndoGalC was verified by detecting the exposure of the terminal N-acetylglucosamine using Griffonia simplicifolia lectin II (GS-II). Paraffin sections (7 µm thick) or isolated cornified cells were incubated with 3.3 µg/ml FITC-labeled GS-IB4 (Sigma) or 20 µg/ml Alexa-Fluor 488-conjugated GS-II (Invitrogen (Molecular probes)) for 30 min at room temperature. Signals were captured with the CCD camera as described above.
Immunohistochemical detection of proliferating cells
Mice were administered 5-bromo-2-deoxyuridine (BrdU, 100 µg/g body weight) (Sigma) 8 days after birth. Two hours later, the mice were killed painlessly and their dorsal skin were mounted in OCT compound (Sakura Finetechnical, Tokyo, Japan) without fixation and immediately frozen in liquid nitrogen. Cryosections (7 µm thick) were treated with 2 N HCl for 20 min and reacted with 0.1% trypsin in calcium- and magnesium-free phosphate-buffered saline for 5 min at 37°C in order to expose the antigenecity. The specimens were incubated with 1/100 diluted anti-BrdU mouse monoclonal antibody (Dako, Glostrup, Denmark) and 1/50 diluted antilaminin rabbit polyclonal antibody (Sigma) overnight at 4°C, and then reacted with second antibodies as above. Nuclei were stained with DAPI. All fluorescent signals were captured as above.
Isolation of cornified cells
Cornified cells were isolated from the dorsal skin of 8-day-old mice by boiling in detergent-containing buffer according to the previous report (Turksen and Troy 2002).
Measurement of moisture content of the skin surface
Moisture levels were measured using moisture checker MY-808S (Scalar, Tokyo, Japan) on the surface of the dorsal skin of EndoGalC TG mice and their sibling WT mice from 7 to 14 days after birth. Individuals were identified by tattoo marks. Measurement was carried out five times for each mouse and the values were averaged. Their body weights were also measured daily.
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Conflict of interest statement |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interest statementReferences |
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None declared.
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Abbreviations |
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3GalT, 1,3galactosyltransferase; Gal epitope, Gal1-3Gal disaccharide; β4GalT, β1,4galactosyltransferase; BrdU, 5-bromo-2-deoxyuridine; CCD, charge-coupled device camera; DAPI, 4',6-diamidino-2-phenylindole dihydrochloride; EndoGalC, Endo-β-galactosidase C; GS-IB4, Griffonia simplicifolia isolectin-IB4; GS-II, Griffonia simplicifolia isolectin-II; TG, transgenic; WT, wild type |
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  R. Uematsu, Y. Shinohara, H. Nakagawa, M. Kurogochi, J.-i. Furukawa, Y. Miura, M. Akiyama, H. Shimizu, and S.-I. Nishimura Glycosylation Specific for Adhesion Molecules in Epidermis and Its Receptor Revealed by Glycoform-focused Reverse Genomics Mol. Cell. Proteomics, February 1, 2009; 8(2): 232 - 244. [Abstract] [Full Text] [PDF]
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