Chang Man Ha

Gyeongsang National University, Shinshū, South Gyeongsang, South Korea

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Publications (17)48.91 Total impact

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    ABSTRACT: Neural epidermal growth factor-like protein-like 2 (NELL2) is a secreted glycoprotein that is predominantly expressed in the nervous system, but little is known about the intracellular movement and secretion mechanism of this protein. By monitoring the localization and movements of enhanced green fluorescent protein (EGFP)-labeled NELL2 in living cultured hippocampal neuroprogenitor HiB5 cells, we determined the subcellular localization of NELL2 and its intracellular movement and secretion mechanism. Cterminal EGFP-fused NELL2 showed a typical expression pattern of secreted proteins, especially with respect to its localization in the endoplasmic reticulum, Golgi apparatus, and punctate structures. Vesicles containing NELL2 exhibited bidirectional movement in HiB5 cells. The majority of the vesicles (70.1%) moved in an anterograde direction with an average velocity of 0.454 μm/s, whereas some vesicles (28.7%) showed retrograde movement with an average velocity of 0.302 μm/s. The movement patterns of NELL2 vesicles were dependent upon the presence of microtubules in HiB5 cells. Anterograde movement of NELL2 did not lead to a detectable accumulation of NELL2 in the peripheral region of the cell, indicating that it was secreted into the culture medium. We also showed that the N-terminal 29 amino acids of NELL2 were important for secretion of this protein. Taken together, these results strongly suggest that the N-terminal region of NELL2 determines both the pattern of its intracellular expression and transport of NELL2 vesicles by high-velocity movement. Therefore, NELL2 may affect the cellular activity of cells in a paracrine or autocrine manner.
    Molecules and Cells 12/2013; 36(6):527-33. · 2.21 Impact Factor
  • Yoonju Kim, Chang Man Ha, Sunghoe Chang
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    ABSTRACT: SNX26, a brain-enriched RhoGAP, plays a key role in dendritic arborization during early neuronal development in the neocortex. In mature neurons, it is localized to dendritic spines, but little is known about its role in later stages of the development. Our results show that SNX26 interacts with PSD-95 in dendritic spines of cultured hippocampal neurons and, as a GAP for Cdc42, it decreased the F-actin content in COS-7 cells and in dendritic spines of neurons. Overexpression of SNX26 resulted in a GAP activity dependent decrease in total protrusions and spine density together with a dramatic inhibition of filopodia-to-spines transformations. Such effects of SNX26 were largely rescued by a constitutively active mutant of Cdc42. Consistently, a shRNA-mediated knockdown of SNX26 significantly increased total protrusions and spine density, resulting in an increase in thin or stubby type spines at the expense of the mushroom spine type. Moreover, endogenous expression of SNX26 was shown to be bi-directionally modulated by neuronal activity. Therefore, we propose that, in addition to its key role in neuronal development, SNX26 also has a role in activity-dependent structural change of dendritic spines in mature neurons.
    Journal of Biological Chemistry 09/2013; · 4.65 Impact Factor
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    ABSTRACT: Calcyon, once known for interacting directly with the dopamine D(1) receptor (D(1)DR), is implicated in various neuropsychiatric disorders including schizophrenia, bipolar disorder, and attention deficit hyperactivity disorder. Although its direct interaction with D(1)DR has been shown to be misinterpreted, it still plays important roles in D(1)DR signaling. Here, we found that calcyon interacts with the PSD-95 and subsequently forms a ternary complex with D(1)DR through PSD-95. Calcyon is phosphorylated on Ser-169 by the PKC activator phorbol 12-myristate 13-acetate or by the D(1)DR agonist SKF-81297, and its phosphorylation increases its association with PSD-95 and recruitment to the cell surface. Interestingly, the internalization of D(1)DR at the cell surface was enhanced by phorbol 12-myristate 13-acetate and SKF-81297 in the presence of calcyon, but not in the presence of its S169A phospho-deficient mutant, suggesting that the phosphorylation of calcyon and the internalization of the surface D(1)DR are tightly correlated. Our results suggest that calcyon regulates D(1)DR trafficking by forming a ternary complex with D(1)DR through PSD-95 and thus possibly linking glutamatergic and dopamine receptor signalings. This also raises the possibility that a novel ternary complex could represent a potential therapeutic target for the modulation of related neuropsychiatric disorders.
    Journal of Biological Chemistry 07/2012; 287(38):31813-22. · 4.65 Impact Factor
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    ABSTRACT: The blood-brain barrier (BBB), which is formed by adherens and tight junctions (TJs) of endothelial cells, maintains homeostasis of the brain. Disrupted intracellular Ca²⁺ homeostasis and breakdown of the BBB have been implicated in the pathogenesis of Alzheimer's disease (AD). The receptor for advanced glycation end products (RAGE) is known to interact with amyloid β-peptide (Aβ) and mediate Aβ transport across the BBB, contributing to the deposition of Aβ in the brain. However, molecular mechanisms underlying Aβ-RAGE interaction-induced alterations in the BBB have not been identified. We found that Aβ₁₋₄₂ induces enhanced permeability, disruption of zonula occludin-1 (ZO-1) expression in the plasma membrane, and increased intracellular calcium and matrix metalloproteinase (MMP) secretion in cultured endothelial cells. Neutralizing antibodies against RAGE and inhibitors of calcineurin and MMPs prevented Aβ₁₋₄₂-induced changes in ZO-1, suggesting that Aβ-RAGE interactions alter TJ proteins through the Ca²⁺-calcineurin pathway. Consistent with these in vitro findings, we found disrupted microvessels near Aβ plaque-deposited areas, elevated RAGE expression, and enhanced MMP secretion in microvessels of the brains of 5XFAD mice, an animal model for AD. We have identified a potential molecular pathway underlying Aβ-RAGE interaction-induced breakage of BBB integrity. This pathway might play an important role in the pathogenesis of AD.
    Journal of Neuroscience 06/2012; 32(26):8845-54. · 6.91 Impact Factor
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    ABSTRACT: NELL2 is a neuron-specific secreted glycoprotein containing an N-terminal thrombospondin I-like domain (TSP-N). In this study, we describe NELL2-Tsp, a novel alternative splice variant of rat NELL2. NELL2-Tsp uses an alternate stop codon resulting in a C-terminal truncated form of NELL2, containing a signal peptide and a TSP-N domain. NELL2-Tsp is a glycosylated protein specifically expressed in brain tissue. NELL2-Tsp and NELL2 are secreted, likely due to the putative signal peptide. However, due to the truncation, the secreted portion of NELL2-Tsp is smaller than that of NELL2. Immunoprecipitation analysis confirmed that NELL2-Tsp was able to associate with NELL2 and with itself. In addition, expression of NELL2-Tsp notably reduced secretion of NELL2 and inhibited NELL2-mediated neurite outgrowth. These results suggest that NELL2-Tsp may act as a negative regulator of wild-type NELL2.
    Biochemical and Biophysical Research Communications 11/2009; 391(1):529-34. · 2.28 Impact Factor
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    ABSTRACT: It was earlier shown that expression of kinesin superfamily-associated protein 3 (KAP3), involved in the neuronal anterograde, microtubule-dependent transport of membrane organelles, increases in the hypothalamus of female rats during the juvenile phase of sexual development. KAP3 mRNA is abundant in the hypothalamus, suggesting that it might be expressed in broadly disseminated neuronal systems controlling neuroendocrine function. The present study identifies one of these systems and provides evidence for an involvement of KAP3 in the excitatory control of female puberty. In situ hybridization and immunohistofluorescence studies revealed that the KAP3 gene is expressed in glutamatergic neurons but not in GABAergic or GnRH neurons. Hypothalamic KAP3 mRNA levels increase during the juvenile period of female prepubertal development, remaining elevated throughout puberty. These changes appear to be, at least in part, estradiol dependent because ovariectomy decreases and estradiol increases KAP3 mRNA abundance. Lowering hypothalamic KAP3 protein levels via intraventricular administration of an antisense oligodeoxynucleotide resulted in reduced release of both glutamate and GnRH from the median eminence and delayed the onset of puberty. The median eminence content of vesicular glutamate transporter 2, a glutamate neuron-selective synaptic protein, and synaptophysin, a synaptic vesicle marker, were also reduced, suggesting that the loss of KAP3 diminishes the anterograde transport of these proteins. Altogether, these results support the view that decreased KAP3 synthesis diminishes GnRH output and delays female sexual development by compromising hypothalamic release of glutamate.
    Endocrinology 09/2008; 149(12):6146-56. · 4.72 Impact Factor
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    ABSTRACT: NELL2, a protein containing EGF-like repeats, is almost exclusively expressed in the nervous system. In the mammalian brain, NELL2 expression is mostly neuronal. NELL2 was previously found to be a secreted protein that functions during embryonic development as a neuronal differentiation and survival factor. We now show that the Nell2 gene is selectively expressed in the two major subtypes of glutamatergic neurons described in the postnatal brain: those containing the vesicular glutamate transporter 1 and those expressing vesicular glutamate transporter 2. No Nell2 mRNA is detected in GABAergic neurons. Likewise, GnRH neurons are devoid of NELL2. During prepubertal development of the female rat, Nell2 mRNA abundance increases selectively in the medial basal hypothalamus, reaching maximal values at the end of the juvenile period, to decline at the time of puberty to intermediate levels. Similar, but less pronounced changes are observed in the preoptic area, but they are absent in the cerebral cortex. A well-established glutamatergic function in the neuroendocrine brain is to enhance release of GnRH, the neurohormone controlling sexual development and the time of puberty. In vivo disruption of NELL2 synthesis via intraventricular administration of antisense oligodeoxynucleotides reduced GnRH release from the medial basal hypothalamus and delayed the initiation of female puberty. These results identify NELL2 as a new component of glutamatergic neurons and provide evidence for its physiological involvement in a major, glutamate-dependent, process of neuroendocrine regulation.
    Neuroendocrinology 07/2008; 88(3):199-211. · 3.54 Impact Factor
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    ABSTRACT: We identified human TRPC3 protein by yeast two-hybrid screening of a human brain cDNA library with human TRPM4b as a bait. Immunoprecipitation and confocal microscopic analyses confirmed the protein-protein interaction between TRPM4b and TRPC3, and these two TRPs were found to be highly colocalized at the plasma membrane of HEK293T cells. Overexpression of TRPM4b suppressed TRPC3-mediated whole cell currents by more than 90% compared to those in TRPC3-expressed HEK293T cells. Furthermore, HEK293T cells stably overexpressing red fluorescent protein (RFP)-TRPM4b exhibited an almost complete abolition of UTP-induced store-operated Ca(2+) entry, which is known to take place via endogenous TRPC channels in HEK293T cells. This study is believed to provide the first clear evidence that TRPM4b interacts physically with TRPC3, a member of a different TRP subfamily, and regulates negatively the channel activity, in turn suppressing store-operated Ca(2+) entry through the TRPC3 channel.
    Biochemical and Biophysical Research Communications 05/2008; 368(3):677-83. · 2.28 Impact Factor
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    ABSTRACT: Thyroid transcription factor 1 (TTF-1) is required for morphogenesis of the fetal diencephalon. Previous reports showed that mice carrying a TTF-1 null mutation lacked normal development of the pituitary gland. In this study, a role for TTF-1 in the regulation of growth hormone and prolactin transcription was identified. In-situ hybridization analysis demonstrated TTF-1 mRNA in the growth hormone-producing cells and prolactin-producing cells of the rat anterior pituitary gland. In the GH3 pituitary cell line, we identified TTF-1 as a factor functionally regulating growth hormone and prolactin transcription. TTF-1 activated prolactin transcription, but inhibited growth hormone transcription. Inhibition and activation of growth hormone and prolactin transcription, respectively, by TTF-1 disappeared upon deletion of the TTF-1 binding motifs within the promoters of these genes. These data suggest that TTF-1 plays a regulatory role in the transcription of growth hormone and prolactin genes and may regulate transdifferentiation of cells expressing these two hormones.
    Biochemical and Biophysical Research Communications 11/2007; 362(1):193-9. · 2.28 Impact Factor
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    ABSTRACT: Munc18, a mammalian homolog of C. elegans Unc, is essential for neurotransmitter release. The aim of this study was to identify estrogen-dependent expression of Munc18-1 and its role in the regulation of glutamate release for puberty onset. Hypothalamic munc18-1 mRNA levels were significantly increased by estrogen treatment in ovariectomized, immature female rats. During pubertal development, the munc18-1 mRNA levels dramatically increased between the juvenile period and the anestrous phase of puberty. Intracerebroventricular administration of an antisense oligodeoxynucleotide against munc18-1 mRNA significantly decreased glutamate release and delayed the day of puberty onset. These results suggest that Munc18-1, expressed in an estrogen-dependent manner, plays an important role in the onset of female puberty via the regulation of glutamate release.
    Molecules and Cells 09/2006; 22(1):30-5. · 2.21 Impact Factor
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    ABSTRACT: In this study we investigated the mRNA expression of NELL2, a neural tissue-specific epidermal growth factor (EGF)-like repeat domain-containing protein, in the developing and adult rat CNS using in situ hybridization histochemistry and northern blot analysis. The possible candidates that interact with or be regulated by NELL2 were screened with a cDNA expression array in antisense (AS) NELL2 oligodeoxynucleotide (ODN)-injected rat hypothalami. NELL2 mRNA was detected as early as embryonic day 10, and was predominant in the CNS throughout the pre-natal stages. Its expression gradually increased during embryonic development and its strong expression was observed throughout the CNS until embryonic day 20. It was detected in the ventricular zone of the spinal cord, medulla and pons in 12-day-old-embryos, suggesting that NELL2 plays a role in the neurogenesis of these areas. After birth its expression gradually decreased, but high levels of expression could be observed in the tenia tecta, piriform cortex, hippocampus, dentate gyrus, cerebellar cortex, ambiguus nucleus, and inferior olivary nucleus of adult rat brains. The analysis of cDNA expression arrays revealed that the administration of AS NELL2 ODN markedly decreased the expression of several Ca2+-binding proteins and those involved in the transport and release of vesicles such as EF-hand Ca2+-binding protein p22 and rab7. This finding was confirmed by relative reverse transcription-polymerase chain reaction. The effect of NELL2 on synaptic vesicle content in median eminence (ME) nerve terminals was determined with synaptophysin levels as a marker protein in the AS NELL2 ODN-injected rat. It was significantly decreased by the AS ODN. These data suggest that NELL2 may play an important role in the development of the CNS as well as maintenance of neural functions, by regulating the intracellular machinery involving Ca2+ signaling, synaptic transport and/or release of vesicles.
    Journal of Neurochemistry 01/2003; 83(6):1389-400. · 3.97 Impact Factor
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    ABSTRACT: We sought to identify genes affected by the aging process in the rat hippocampus using cDNA expression array analysis. RNA samples were extracted from the hippocampus of 2-month-old and 20-month-old rats and reverse-transcribed in the presence of [32P]dCTP. Membrane sets of Rat Atlas array 2 (Clontech) were hybridized with cDNA probe sets. Among a total of 1176 cDNAs, 23 showed significant (more than 2-fold) changes between groups. Eight genes were increased in the old group, while the remaining fifteen genes were decreased. Reverse transcription-polymerase chain reaction (RT-PCR) was used to validate the relative expression pattern obtained by the cDNA array. The results were consistent for 20 of the 23 genes tested. Most interesting findings were the decrease in expression of proteins for energy metabolism, proteins involved in secretion, and ribosomal proteins. The possible physiological significance of these changes are discussed.
    Neuroreport 04/2002; 13(3):285-9. · 1.40 Impact Factor
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    ABSTRACT: To identify estrogen (E)-responsive genes that may play important roles in the sexual differentiation and maturation of the neuroendocrine hypothalamus, we used mRNA differential display PCR to analyze hypothalamic RNA derived from estrogen-sterilized rats (ESRs). Neonatal rats were s.c.-injected with 100 μg of 17β-estradiol-benzoate (EB) for 5 days. Approximately 300 out of more than 2000 RNAs examined displayed a differential expression pattern between hypothalami of the ESR females compared to their 60-day-old controls. EB-dependent expression of these genes was further analyzed by low-density cDNA array using cDNA probe sets reverse-transcribed from the same groups; 98 genes were confirmed to be differentially expressed. We selected 41 clones that showed higher density differences between the two probe sets than mean density difference in control cyclophilin cDNA blots in the cDNA array. After being cloned into pGEM-T vectors, their sequences were analyzed. Homology searches identified four genes as a protein kinase C (PKC)-binding protein, NELL2 (clone 6-1), a thyroid nuclear factor, TTF-1 (9-1), Munc18-1 (17-6), and leuserpin-2 (18-5). The other 22 genes were similar to reported genes or cDNAs such as mouse kinesin-associated protein 3 (KAP3, 8b), mouse IgE binding lectin (15-1), normalized rat brain cDNA (5-1), rat cDNA (8-1) and rat embryonic cDNA (17-1). Fifteen clones such as clone 7-3 showed no match in the GenBank Database. Further characterization of eight clones (17-1, 7-3, 8-1, 5-1, NELL2, KAP3 homolog, IgE binding lectin homolog, and TTF-1) showed that their expression in the adult female rat hypothalamus is sensitive to neonatal treatment with EB. They showed brain-specific expression and moreover, showed an increase in their mRNA level before the initiation of puberty. Some of them showed gender differences in their different postnatal expression pattern. We speculate that further study will demonstrate that many of the E-regulated genes identified in the present study play important roles in the regulation of the sexual differentiation and E-dependent maturation of the hypothalamus.
    Molecular Brain Research 01/2002; · 2.00 Impact Factor
  • M S Kim, C M Ha, B J Lee
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    ABSTRACT: The ribonuclease (RNase) protection assay (RPA) is an extremely sensitive technique used to determine specific mRNAs from cell and tissue extracts. The present protocol presents detailed procedures for a conventional RPA using antisense RNA probes purified with a Fullengther apparatus. The Fullengther has the advantage of being a relatively quick and safe procedure compared to more conventional methods for purification of full-length RNA probes. Using this protocol, we sought to simultaneously determine multiple mRNA species, including splice variants of the type I receptor (PAC(1)) of pituitary adenylate cyclase-activating polypeptide (PACAP), an important mediator in the regulation of luteinizing hormone-releasing hormone (LHRH) synthesis by ovarian steroids such as progesterone [7]. PAC(1) has more than eight splice variants. We have been able to discriminate the hop1 variant from other splice variants. To improve our understanding of the regulation mechanism of genes that are related to each other, such as LHRH and PACAP, it is most important to simultaneously determine genes that are involved in the same physiological areas of regulation. Using only 5 microg of total RNA sample from a single rat preoptic area, we simultaneously determined five different transcripts, including four rare mRNA species such as LHRH, PACAP, and hop1 variant and other splice variants of PAC(1), as well as the internal control of cyclophilin mRNA. This protocol provides a method for the simultaneous determination of multiple transcripts using the RPA.
    Brain Research Protocols 08/2001; 7(3):277-85. · 1.82 Impact Factor
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    ABSTRACT: Central administration of an antisense oligodeoxynucleotide against type I pituitary adenylate cyclase-activating polypeptide receptor suppresses synthetic activities of LHRH-LH axis during the pubertal process In the present study, we determined the expression of pituitary adenylate cyclase-activating polypeptide (PACAP) and PACAP receptor type I (PAC1) genes during juvenile development and the pubertal process. Female rats were assigned--based on uterine weights, the presence and abundance of uterine fluid, and their vaginal patency--to one of the following: anestrus (AE), early proestrus (EP), late proestrus (LP) or first estrus (E). The hypothalami from 22-, 24- and 26-day-old animals and from those in the peripubertal phases of AE, EP, LP and E were collected, and the content of PACAP and PAC1 mRNA was assessed. These levels were found to decrease in EP and LP. To determine the effect of PACAP on prepubertal luteinizing hormone-releasing hormone (LHRH) and LH synthesis through PAC1, a PAC1 antisense oligodeoxynucleotide (ODN) was i.c.v.-administered, and mRNA levels of LHRH, LH beta, and LHRH receptor (LHRH-R) were determined. Prepubertal increases in LHRH, LH beta, and LHRH-R mRNA levels were markedly suppressed, and the onset of puberty was delayed by the i.c.v. injection of the antisense PAC1 ODN. These data suggest that PACAP may play a role in the regulation of hypothalamic LHRH neurons, through which it regulates synthetic machinery of pituitary LH, during the pubertal process.
    Molecular Brain Research 09/2000; 80(1):35-45. · 2.00 Impact Factor
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    ABSTRACT: Pituitary adenylate cyclase-activating polypeptide (PACAP) regulates pituitary hormone biosynthesis and secretion through its cognate receptors. PACAP also plays an important role in the regulation of ovarian steroid biosynthesis. If so, there might be a feedback regulation of hypothalamic PACAP synthesis by the pituitary and by ovarian steroids. In the present study, we used RNase protection assays to determine changes in mRNA levels of PACAP and type I PACAP receptor (PAC(1)) under the conditions of ovariectomy and replacement with ovarian steroids. Progesterone (P) alone or in combination with estradiol (E) induced significant increases in PACAP mRNA level in the medial basal hypothalamus (MBH) and PAC(1) mRNA levels in MBH and the preoptic area (POA). This finding suggests that feedback regulation takes place between the ovary and hypothalamic PACAP neurons. P is known to be a major regulatory feedback factor for hypothalamic luteinizing hormone-releasing hormone (LHRH) neurons, but P receptor is not present in these neurons. Therefore, we examined a possible involvement of PACAP in the feedback regulatory pathway of P to LHRH neurons. After an antisense PAC(1) oligodeoxynucleotide (ODN) was i.c.v.-injected into the third ventricle of E and P-treated rats, LHRH mRNA levels were determined. The ODN markedly decreased the P-induced increase in the LHRH mRNA level. Taken together, the present data suggest that PACAP may play a role as a mediator in the regulation of LHRH synthetic machinery by stimulatory feedback of P.
    Molecular Brain Research 06/2000; 78(1-2):59-68. · 2.00 Impact Factor
  • Chang Man Ha, Hae Mook Kang, Byung Ju Lee
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    ABSTRACT: In the present study we determined if NELL2, a neural tissue‐specific protein containing 6 epidermal growth factor (EGF)‐like repeat domains, plays an important role in the regulation of puberty initiation in the rat hypothalamus. We originally found that NELL2 is a new estrogen‐responsive gene in hypothalami derived from estrogen‐sterilized and control rats using a PCR differential display. In the 40‐day‐old female rat hypothalamus, NELL2 was up‐regulated by neonatal estrogen treatment. In situ hybridization his to chemistry showed that NELL2 is very abundant in the ventromedial hypothalamic nucleus that is responsible for the control of sex behavior. NELL2 mRNA level in the medial basal hypothalamus showed a dramatic increase before female puberty onset, which suggests that NELL2 may be involved in the process regulating female puberty onset. We attemped to block NELL2 synthesis with intrace rebroventricular injection of an antisense oligodeoxynucleotide (ODN) to the NELL2 mRNA, and examined its effect on the puberty onset of the female rat. The antisense ODN significantly delayed puberty initiation determined by vaginal opening. In summary, NELL2 may play an important role in the regulation of female puberty onset.

Publication Stats

154 Citations
48.91 Total Impact Points

Institutions

  • 2013
    • Gyeongsang National University
      Shinshū, South Gyeongsang, South Korea
  • 2009
    • Yonsei University Hospital
      Sŏul, Seoul, South Korea
  • 2000–2008
    • University of Ulsan
      • Department of Biological Sciences
      Ulsan, Ulsan, South Korea