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Expression of androgen receptor, estrogen receptor α and β in the dermal papilla of human hair follicles in vivo
Journal of Dermatological Science (2004) 36, 176—179
www.intl.elsevierhealth.com/journals/jods
LETTER TO THE EDITOR
Expression of androgen receptor, estrogen receptor a and b in the dermal papilla of human hair follicles in vivo Sir, Hair growth and cycles are under the influence of sex hormones like androgens and estrogens, and dermal papilla cells (DPCs) are known to be the target of hair growth regulation by these hormones, which affect the transcription of modulatory signals in these cells [1]. Androgens act after binding to the androgen receptor (AR), a member of steroid hormone nuclear receptor superfamily, and DPCs cultured from androgen-sensitive balding frontal scalp were found to contain higher levels of AR than DPCs from androgen-insensitive occipital scalp areas [2]. Estrogen also appears to be able to influence hair growth. Topical estrogen significantly accelerated hair regrowth after chemotherapy-induced alopecia in an established murine model [3]. Estrogens act via two known receptors denoted a (ERa) and b (ERb). Recently, these two receptors were found to display different immunohistochemical localization profiles in human non-balding occipital scalp skin, where ERb is predominantly expressed, suggesting their different functional roles in cutaneous biology [4]. The aim of this study was to investigate whether there is a difference in the expression of AR, ERa and ERb in DPCs of the balding vertex (VERTEXDPCs) and of non-balding occipital scalp skin (OCCIPUTDPCs). Scalp tissue samples (1 2—2.5 cm) were obtained from the androgen-dependent vertex and androgen-independent occipital scalps of the 8 male volunteers aged from 22 to 35 years (Hamilton classification II—IV) after subjects had provided written informed consent. For semiquantitative RTPCR and Western blot analysis, dermal papillae (DPs) from defined scalp sites were plucked from anagen hair follicles as suggested previously [5]. Approximately 130—150 DPs were obtained from each area,
respectively. To detect mRNAs expression, total RNA was extracted from DP homogenates using Trizol reagent (Gibco BRL, Germany) and was reverse transcribed. PCR amplification was performed using each specific primer (AR: sense, 50 -CCTGGCTTCCGCAAC TTACAC-30 and antisense, 50 -GGACTTGTGCATGCGGTACTCA-30 ; ERa: sense, 50 -GTACAGCATGAAGTGCAAGA-30 and antisense, 50 -AAGGAATGCGATGAAGTA GA-30 ; ERb: sense, 50 -GACATTCTATAGCCCTGCTG-30 and antisense, 50 -TCCGCATACAGATGTGATAA-30 ) (RT-PCR). The protein expression of AR, ERa and ERb was determined by immunoblotting using primary polyclonal rabbit anti-human AR (SC816), anti-human ERa (SC544) and ERb (SC8974) from Santa Cruz Biotechnology, California, and a horseradish peroxidase-conjugate goat anti-rabbit secondary antibody. The expression was detected using the ECL system (Amersham Pharmacia Biotech, Little Chalfont, UK) and analyzed using a Bio-Rad GS-700 imaging densitometer (Hercules, CA). The rest small portions were fixed into formalin, embedded in paraffin and sliced serially. To detect the immunoreactivity of receptors sections were incubated with mouse anti-human AR (M3562, Dako, Carpinteria, USA) and the above ER antibodies according to the manufacturer’s recommendations. To identify differences in the transcription of AR in defined two sites, their mRNA levels were examined. In all samples, mean mRNAs of AR were 3.4fold higher in VERTEXDPCs than those from occipital scalp (Fig. 1A). These site-specific different expressions of AR were consistent in Western blot (Fig. 1B). VERTEX DPCs showed 1.9-fold higher AR expression than their counterparts, consistent with the results of cultured DPCs from hair follicles from the frontal and occipital scalp [2]. Immunohistochemical study showed that AR was localized to the nuclei of the VERTEX DPCs at the anagen stage (six of eight samples), although not all the DPCs were positive (Fig. 1C). In sebaceous gland, AR immunoreactivity was mostly confined to the nuclei of the basal cells as previously described (Fig. 1D) [4].
0923-1811/$30.00 # 2004 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jdermsci.2004.09.004
Letter to the Editor
Fig. 1 The expressions of mRNA, protein and AR immunoreactivity on the DPCs of hair follicles from human male vertex and occipital scalp skin. (A) RT-PCR analysis of AR mRNA expression in DP homogenates from two sites. (B) Western blot analysis of AR protein expression in DP homogenates from defined sites. The bands of RT-PCR and Western blot analyses are representative results from two sites. Results shown are averages S.E.M. from sets of four independent volunteers, respectively. The AR mRNA and protein levels in VERTEXDPCs are expressed relative to those of the occipital scalp as a control. Experiments were performed in triplicate. *P < 0.05 using Wilcoxon signed rank test with SAS statistical software (SAS Institute Inc., Cary, NC) (A and B). (C) Immunoreactivity for AR on follicular DPCs of vertex scalp. The expression of AR was localized to nuclei of DPCs (arrow head), though not all DPCs were positive. Some of DPCs were negative for the AR. (D) Immunohistochemical localization of AR to the sebaceous gland as an internal positive control. Staining for AR was mostly restricted to the basal cells of sebaceous gland. Original magnifications, 200 (C, D). Images were obtained from same 31-year-old volunteer.
Reversely, ERa mRNA expression levels were 2.2fold higher in OCCIPUTDPCs than in their counterparts. ERb mRNA was consistently highly expressed by 4.2fold in OCCIPUTDPCs versus VERTEXDPCs (Fig. 2A). ERa protein was consistently elevated in OCCIPUTDPCs by 1.8-fold higher versus vertex and ERb was also definitely increased in DPCs from non-balding
177
Fig. 2 The expressions of mRNA, protein and immunoreactivity of ERa and ERb on the DPCs of hair follicles from human male vertex and occipital scalp skin. (A) RT-PCR analysis of ERa and ERb mRNA expression in DP homogenates from two sites. (B) Western blot analysis of ERa and ERb protein expression in DP homogenates from defined sites. The bands of RT-PCR and Western blot analyses are representative results from two sites. Results shown are mean densities S.E.M. from sets of four independent volunteers, respectively. The ERa and ERb mRNA and protein levels in OCCIPUTDPCs are expressed relative to those of the vertex scalp as a control. Experiments were performed in triplicate. * P < 0.05 using Wilcoxon signed rank test with SAS statistical software (SAS Institute Inc., Cary, NC) (A and B). (C) Immunoreactivity for ERa on DPCs of hair follicle from human male occipital scalp. The expression of ERa was localized to a small number of nuclei of DPCs (arrow head). (D) Immunohistochemical localization of ERa to the sebaceous gland as an internal positive control. Staining for ERa was diffusely shown in basal cells and partly differentiated sebocytes, not restricted to the nucleus. (E) Immunohistochemical localization of ERb to the follicular DP of occipital scalp. Specific nuclear staining was shown in the some of DPCs (arrow head). (F) The immunoreactivity of ERb in the sebaceous gland. The
178
scalps, with a 1.8-fold stronger intensity (Fig. 2B). The ERa immunoreactivity was visualized in nuclei of a few DPCs of occipital scalp (three of eight samples) (Fig. 2C) and strongly observed on the basal cells and differentiating sebocytes of sebaceous gland (Fig. 2D). A specific nuclear staining for ERb was shown in the several OCCIPUTDPCs (Fig. 2E). In sebaceous gland, ERb was widely expressed on the basal and differentiating sebocytes with both cytoplasmic and nuclear staining (Fig. 2F). We demonstrate here the in vivo complementary expression patterns of AR, ERa and b in DPCs from the human vertex and occipital scalp. The different sensitivity of hair follicles to androgens could be attributed in part to their dissimilar expression AR levels, although diverse molecular machineries consisted of the downstream AR coactivators and coregulators may be extensively involved in the regulation of the androgen effect in the organs [6]. Recently cloned ERb is high homologue of ERa, but shares only 55% sequence homology in the ligand binding domains, and after ligand binding they both form homodimers and heterodimers, which interact with hormone response elements and contribute to differential responses. Most recently, the two different receptors were reported to express in human hair follicle in vivo [7]. Tissue specific distributions may also contribute to variable responsiveness [8]. ERa and ERb demonstrated different immunohistochemical localization profiles in human skin and in cultured DPCs, where they showed different expressional patterns, i.e., ERb showed strong nuclear expression, and ERa, granular diffuse cytoplasmic staining [4]. A stable and specific interaction between AR and ERa was suggested to have potential physiological ramifications. AR and ERa were reported to interact directly and to downregulate transactivation each other. On the other hand, ERb was demonstrated not to interact with AR or to modulate the transcriptional activity of AR. Thus ERb is unlikely to be affected by high levels of AR expression, and thus it was suggested to control estrogen signaling in organs and systems where containing all three receptors [9]. In summary, we demonstrate the differential expressions of AR, ERa and ERb in the DPCs of hair follicles from vertex and occipital scalp. Our results indicate and further support the idea that the hair
ERb was widely expressed to the nuclei and cytoplasms of basal cells and sebocytes Original magnifications, 200 (C—F). Images were obtained from same 31-year-old volunteer.
Letter to the Editor
follicle is a sex hormone regulated organ with genetically determined site-specific response differences. Further studies of the functional interactions of receptors in human hair follicles are warranted to promote our understanding of the effects of sex hormones on hair growth.
Acknowledgments This study was supported by a grant of the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea (03-PJ1-PG1-CH130001). This work was presented as a poster at 11th annual meeting of the SHSR joint with KHRS at November 29, 2003, Jeju, Korea. The authors want to thank Min Jeong Lee, Clinical Research Institute at Seoul National University Hospital, Seoul, Korea for valuable assistance with the statistical evaluation.
References [1] Paus R, Cotsarelis G. The biology of hair follicles. N Engl J Med 1999;341:491—7. [2] Hibberts NA, Howell AE, Randall VA. Balding hair follicle dermal papilla cells contain higher levels of androgen receptors than those from non-balding scalp. J Endocrinol 1998;156:59—65. [3] Ohnemus U, Unalan M, Handjiski B, Paus R. Topical estrogen accelerates hair regrowth in mice after chemotherapy-induced alopecia by favoring the dystrophic catagen response pathway to damage. J Invest Dermatol 2004; 122:7—13. [4] Thornton MJ, Nelson LD, Taylor AH, Mulligan K, Al-Azzawi F, Messenger AG. Human non-balding scalp dermal papilla cells express estrogen receptor beta (ERb) protein in vivo and in vitro. In: Van Neste D, editor. Hair science and technology. Tournai: Skinterface; 2003. p. 105—20. [5] Asada Y, Sonoda T, Ojiro M, Kurata S, Sato T, Ezaki T, et al. 5 Alpha-reductase type 2 is constitutively expressed in the dermal papilla and connective tissue sheath of the hair follicle in vivo but not during culture in vitro. J Clin Endocrinol Metab 2001;86:2875—80. [6] Lee DK, Chang C. Endocrine mechanisms of disease: expression and degradation of androgen receptor: mechanism and clinical implication. J Clin Endocrinol Metab 2003;88:4043— 54. [7] Nelson LD, Messenger AG, Taylor AH, Bharkhada RJ, Laing I, Thornton MJ. Evidence for a glucocorticoid-dependent mechanism for the selective action of estradiol via estrogen receptor beta in cultured human scalp dermal papilla cells. JDDG 2004;2:508 [abstract]. [8] Taylor AH, Al-Azzawi F. Immunolocalisation of oestrogen receptor beta in human tissues. J Mol Endocrinol 2000; 24:145—55. [9] Panet-Raymond V, Gottlieb B, Beitel LK, Pinsky L, Trifiro MA. Interactions between androgen and estrogen receptors and the effects on their transactivational properties. Mol Cell Endocrinol 2000;167:139—50.
Letter to the Editor
179
Oh Sang Kwon Ji Hyun Han Hyeon Gyeong Yoo Se Rah Lee Kyu Han Kim Hee Chul Eun Kwang Hyun Cho Department of Dermatology Seoul National University College of Medicine Laboratory of Cutaneous Aging and Hair Biology Research Clinical Research Institute
Seoul National University Hospital Yongon-Dong 28, Chongno-Gu 110-744, Seoul Republic of Korea Young Chul Sim AmorePacific R&D Center Yongin-Si, Gyeonggi-Do Republic of Korea
Corresponding author. Tel.: +82 2 2072 2412 fax: +82 2 742 7344 E-mail address: [email protected] (K.H. Cho) 10 July 2004
www.intl.elsevierhealth.com/journals/jods
LETTER TO THE EDITOR
Expression of androgen receptor, estrogen receptor a and b in the dermal papilla of human hair follicles in vivo Sir, Hair growth and cycles are under the influence of sex hormones like androgens and estrogens, and dermal papilla cells (DPCs) are known to be the target of hair growth regulation by these hormones, which affect the transcription of modulatory signals in these cells [1]. Androgens act after binding to the androgen receptor (AR), a member of steroid hormone nuclear receptor superfamily, and DPCs cultured from androgen-sensitive balding frontal scalp were found to contain higher levels of AR than DPCs from androgen-insensitive occipital scalp areas [2]. Estrogen also appears to be able to influence hair growth. Topical estrogen significantly accelerated hair regrowth after chemotherapy-induced alopecia in an established murine model [3]. Estrogens act via two known receptors denoted a (ERa) and b (ERb). Recently, these two receptors were found to display different immunohistochemical localization profiles in human non-balding occipital scalp skin, where ERb is predominantly expressed, suggesting their different functional roles in cutaneous biology [4]. The aim of this study was to investigate whether there is a difference in the expression of AR, ERa and ERb in DPCs of the balding vertex (VERTEXDPCs) and of non-balding occipital scalp skin (OCCIPUTDPCs). Scalp tissue samples (1 2—2.5 cm) were obtained from the androgen-dependent vertex and androgen-independent occipital scalps of the 8 male volunteers aged from 22 to 35 years (Hamilton classification II—IV) after subjects had provided written informed consent. For semiquantitative RTPCR and Western blot analysis, dermal papillae (DPs) from defined scalp sites were plucked from anagen hair follicles as suggested previously [5]. Approximately 130—150 DPs were obtained from each area,
respectively. To detect mRNAs expression, total RNA was extracted from DP homogenates using Trizol reagent (Gibco BRL, Germany) and was reverse transcribed. PCR amplification was performed using each specific primer (AR: sense, 50 -CCTGGCTTCCGCAAC TTACAC-30 and antisense, 50 -GGACTTGTGCATGCGGTACTCA-30 ; ERa: sense, 50 -GTACAGCATGAAGTGCAAGA-30 and antisense, 50 -AAGGAATGCGATGAAGTA GA-30 ; ERb: sense, 50 -GACATTCTATAGCCCTGCTG-30 and antisense, 50 -TCCGCATACAGATGTGATAA-30 ) (RT-PCR). The protein expression of AR, ERa and ERb was determined by immunoblotting using primary polyclonal rabbit anti-human AR (SC816), anti-human ERa (SC544) and ERb (SC8974) from Santa Cruz Biotechnology, California, and a horseradish peroxidase-conjugate goat anti-rabbit secondary antibody. The expression was detected using the ECL system (Amersham Pharmacia Biotech, Little Chalfont, UK) and analyzed using a Bio-Rad GS-700 imaging densitometer (Hercules, CA). The rest small portions were fixed into formalin, embedded in paraffin and sliced serially. To detect the immunoreactivity of receptors sections were incubated with mouse anti-human AR (M3562, Dako, Carpinteria, USA) and the above ER antibodies according to the manufacturer’s recommendations. To identify differences in the transcription of AR in defined two sites, their mRNA levels were examined. In all samples, mean mRNAs of AR were 3.4fold higher in VERTEXDPCs than those from occipital scalp (Fig. 1A). These site-specific different expressions of AR were consistent in Western blot (Fig. 1B). VERTEX DPCs showed 1.9-fold higher AR expression than their counterparts, consistent with the results of cultured DPCs from hair follicles from the frontal and occipital scalp [2]. Immunohistochemical study showed that AR was localized to the nuclei of the VERTEX DPCs at the anagen stage (six of eight samples), although not all the DPCs were positive (Fig. 1C). In sebaceous gland, AR immunoreactivity was mostly confined to the nuclei of the basal cells as previously described (Fig. 1D) [4].
0923-1811/$30.00 # 2004 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jdermsci.2004.09.004
Letter to the Editor
Fig. 1 The expressions of mRNA, protein and AR immunoreactivity on the DPCs of hair follicles from human male vertex and occipital scalp skin. (A) RT-PCR analysis of AR mRNA expression in DP homogenates from two sites. (B) Western blot analysis of AR protein expression in DP homogenates from defined sites. The bands of RT-PCR and Western blot analyses are representative results from two sites. Results shown are averages S.E.M. from sets of four independent volunteers, respectively. The AR mRNA and protein levels in VERTEXDPCs are expressed relative to those of the occipital scalp as a control. Experiments were performed in triplicate. *P < 0.05 using Wilcoxon signed rank test with SAS statistical software (SAS Institute Inc., Cary, NC) (A and B). (C) Immunoreactivity for AR on follicular DPCs of vertex scalp. The expression of AR was localized to nuclei of DPCs (arrow head), though not all DPCs were positive. Some of DPCs were negative for the AR. (D) Immunohistochemical localization of AR to the sebaceous gland as an internal positive control. Staining for AR was mostly restricted to the basal cells of sebaceous gland. Original magnifications, 200 (C, D). Images were obtained from same 31-year-old volunteer.
Reversely, ERa mRNA expression levels were 2.2fold higher in OCCIPUTDPCs than in their counterparts. ERb mRNA was consistently highly expressed by 4.2fold in OCCIPUTDPCs versus VERTEXDPCs (Fig. 2A). ERa protein was consistently elevated in OCCIPUTDPCs by 1.8-fold higher versus vertex and ERb was also definitely increased in DPCs from non-balding
177
Fig. 2 The expressions of mRNA, protein and immunoreactivity of ERa and ERb on the DPCs of hair follicles from human male vertex and occipital scalp skin. (A) RT-PCR analysis of ERa and ERb mRNA expression in DP homogenates from two sites. (B) Western blot analysis of ERa and ERb protein expression in DP homogenates from defined sites. The bands of RT-PCR and Western blot analyses are representative results from two sites. Results shown are mean densities S.E.M. from sets of four independent volunteers, respectively. The ERa and ERb mRNA and protein levels in OCCIPUTDPCs are expressed relative to those of the vertex scalp as a control. Experiments were performed in triplicate. * P < 0.05 using Wilcoxon signed rank test with SAS statistical software (SAS Institute Inc., Cary, NC) (A and B). (C) Immunoreactivity for ERa on DPCs of hair follicle from human male occipital scalp. The expression of ERa was localized to a small number of nuclei of DPCs (arrow head). (D) Immunohistochemical localization of ERa to the sebaceous gland as an internal positive control. Staining for ERa was diffusely shown in basal cells and partly differentiated sebocytes, not restricted to the nucleus. (E) Immunohistochemical localization of ERb to the follicular DP of occipital scalp. Specific nuclear staining was shown in the some of DPCs (arrow head). (F) The immunoreactivity of ERb in the sebaceous gland. The
178
scalps, with a 1.8-fold stronger intensity (Fig. 2B). The ERa immunoreactivity was visualized in nuclei of a few DPCs of occipital scalp (three of eight samples) (Fig. 2C) and strongly observed on the basal cells and differentiating sebocytes of sebaceous gland (Fig. 2D). A specific nuclear staining for ERb was shown in the several OCCIPUTDPCs (Fig. 2E). In sebaceous gland, ERb was widely expressed on the basal and differentiating sebocytes with both cytoplasmic and nuclear staining (Fig. 2F). We demonstrate here the in vivo complementary expression patterns of AR, ERa and b in DPCs from the human vertex and occipital scalp. The different sensitivity of hair follicles to androgens could be attributed in part to their dissimilar expression AR levels, although diverse molecular machineries consisted of the downstream AR coactivators and coregulators may be extensively involved in the regulation of the androgen effect in the organs [6]. Recently cloned ERb is high homologue of ERa, but shares only 55% sequence homology in the ligand binding domains, and after ligand binding they both form homodimers and heterodimers, which interact with hormone response elements and contribute to differential responses. Most recently, the two different receptors were reported to express in human hair follicle in vivo [7]. Tissue specific distributions may also contribute to variable responsiveness [8]. ERa and ERb demonstrated different immunohistochemical localization profiles in human skin and in cultured DPCs, where they showed different expressional patterns, i.e., ERb showed strong nuclear expression, and ERa, granular diffuse cytoplasmic staining [4]. A stable and specific interaction between AR and ERa was suggested to have potential physiological ramifications. AR and ERa were reported to interact directly and to downregulate transactivation each other. On the other hand, ERb was demonstrated not to interact with AR or to modulate the transcriptional activity of AR. Thus ERb is unlikely to be affected by high levels of AR expression, and thus it was suggested to control estrogen signaling in organs and systems where containing all three receptors [9]. In summary, we demonstrate the differential expressions of AR, ERa and ERb in the DPCs of hair follicles from vertex and occipital scalp. Our results indicate and further support the idea that the hair
ERb was widely expressed to the nuclei and cytoplasms of basal cells and sebocytes Original magnifications, 200 (C—F). Images were obtained from same 31-year-old volunteer.
Letter to the Editor
follicle is a sex hormone regulated organ with genetically determined site-specific response differences. Further studies of the functional interactions of receptors in human hair follicles are warranted to promote our understanding of the effects of sex hormones on hair growth.
Acknowledgments This study was supported by a grant of the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea (03-PJ1-PG1-CH130001). This work was presented as a poster at 11th annual meeting of the SHSR joint with KHRS at November 29, 2003, Jeju, Korea. The authors want to thank Min Jeong Lee, Clinical Research Institute at Seoul National University Hospital, Seoul, Korea for valuable assistance with the statistical evaluation.
References [1] Paus R, Cotsarelis G. The biology of hair follicles. N Engl J Med 1999;341:491—7. [2] Hibberts NA, Howell AE, Randall VA. Balding hair follicle dermal papilla cells contain higher levels of androgen receptors than those from non-balding scalp. J Endocrinol 1998;156:59—65. [3] Ohnemus U, Unalan M, Handjiski B, Paus R. Topical estrogen accelerates hair regrowth in mice after chemotherapy-induced alopecia by favoring the dystrophic catagen response pathway to damage. J Invest Dermatol 2004; 122:7—13. [4] Thornton MJ, Nelson LD, Taylor AH, Mulligan K, Al-Azzawi F, Messenger AG. Human non-balding scalp dermal papilla cells express estrogen receptor beta (ERb) protein in vivo and in vitro. In: Van Neste D, editor. Hair science and technology. Tournai: Skinterface; 2003. p. 105—20. [5] Asada Y, Sonoda T, Ojiro M, Kurata S, Sato T, Ezaki T, et al. 5 Alpha-reductase type 2 is constitutively expressed in the dermal papilla and connective tissue sheath of the hair follicle in vivo but not during culture in vitro. J Clin Endocrinol Metab 2001;86:2875—80. [6] Lee DK, Chang C. Endocrine mechanisms of disease: expression and degradation of androgen receptor: mechanism and clinical implication. J Clin Endocrinol Metab 2003;88:4043— 54. [7] Nelson LD, Messenger AG, Taylor AH, Bharkhada RJ, Laing I, Thornton MJ. Evidence for a glucocorticoid-dependent mechanism for the selective action of estradiol via estrogen receptor beta in cultured human scalp dermal papilla cells. JDDG 2004;2:508 [abstract]. [8] Taylor AH, Al-Azzawi F. Immunolocalisation of oestrogen receptor beta in human tissues. J Mol Endocrinol 2000; 24:145—55. [9] Panet-Raymond V, Gottlieb B, Beitel LK, Pinsky L, Trifiro MA. Interactions between androgen and estrogen receptors and the effects on their transactivational properties. Mol Cell Endocrinol 2000;167:139—50.
Letter to the Editor
179
Oh Sang Kwon Ji Hyun Han Hyeon Gyeong Yoo Se Rah Lee Kyu Han Kim Hee Chul Eun Kwang Hyun Cho Department of Dermatology Seoul National University College of Medicine Laboratory of Cutaneous Aging and Hair Biology Research Clinical Research Institute
Seoul National University Hospital Yongon-Dong 28, Chongno-Gu 110-744, Seoul Republic of Korea Young Chul Sim AmorePacific R&D Center Yongin-Si, Gyeonggi-Do Republic of Korea
Corresponding author. Tel.: +82 2 2072 2412 fax: +82 2 742 7344 E-mail address: [email protected] (K.H. Cho) 10 July 2004