The Non-Canonical Effect of N-Acetyl-D-Glucosamine Kinase on the Formation of Neuronal Dendrites

Lee, HyunSook,Cho, Sun-Jung,Moon, Il Soo Korean Society for Molecular and Cellular Biology 2014 Molecules and cells Vol.37 No.3

N-acetylglucosamine kinase (GlcNAc kinase or NAGK; EC 2.7.1.59) is a N-acetylhexosamine kinase that belong to the sugar kinase/heat shock protein 70/actin superfamily. In this study, we investigated both the expression and function of NAGK in neurons. Immunohistochemistry of rat brain sections showed that NAGK was expressed at high levels in neurons but at low levels in astrocytes. Immunocytochemistry of rat hippocampal dissociate cultures confirmed these findings and showed that NAGK was also expressed at low levels in oligodendrocytes. Furthermore, several NAGK clusters were observed in the nucleoplasm of both neuron and glia. The overexpression of EGFP- or RFP (DsRed2)-tagged NAGK in rat hippocampal neurons (DIV 5-9) increased the complexity of dendritic architecture by increasing the numbers of primary dendrites and dendritic branches. In contrast, knockdown of NAGK by shRNA resulted in dendrite degeneration, and this was prevented by the co-expression of RFP-tagged NAGK. These results suggest that the upregulation of dendritic complexity is a non-canonical function of NAGK.

Preparation of a Monoclonal Antibody against Gintonin and Its Use in an Enzyme Immunoassay.

Lee, Byung-Hwan,Choi, Sun-Hye,Kim, Hyeon-Joong,Jung, Seok-Won,Kim, Hyunsook,Shin, Ho-Chul,Lee, Joon-Hee,Hwang, Sung-Hee,Kim, Hyoung-Chun,Nah, Seung-Yeol Pharmaceutical Society of Japan 2015 Biological & pharmaceutical bulletin Vol.38 No.10

<P>Gintonin is a novel ginseng-derived G protein-coupled lysophosphatidic acid (LPA) receptor ligand. Gintonin elicits an [Ca(2+)]i transient in animal cells via activation of LPA receptors. In vitro studies have shown that gintonin regulates various calcium-dependent ion channels and receptors. In in vivo studies, gintonin elicits anti-Alzheimer's disease activity through the activation of the non-amyloidogenic pathway and anti-metastatic effects through the inhibition of autotaxin. However, a method for gintonin quantitation in ginseng has not been developed. In the present study, we developed an enzyme immunoassay (EIA) to measure gintonin. A monoclonal antibody was raised in a mouse using gintonin as the immunogen, and an indirect competitive EIA was used to measure gintonin. The working range was 0.01-10???µg per assay. The anti-gintonin monoclonal antibody did not cross-react with the ginsenosides Ra, Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, and Rg3 or with LPAs such as LPA C16:0, LPA C18:0, LPA C18:1, and LPA C18:2. Using a standard curve, we measured the amount of gintonin in various ginseng extract fractions. Interestingly, we only detected a little amount of gintonin in conventional hot water extracts of Korean red ginseng. However, we can measure gintonin after ethanol extraction of Korean red ginseng marc. Thus, gintonin can be extracted from ginseng with ethanol but not water, and the remaining Korean red ginseng marc can be used to obtain gintonin. These results indicate that the EIA with the anti-gintonin monoclonal antibody can be used to quantify gintonin in various ginseng preparations, including commercial ginseng products.</P>

How Chromosome Mis-Segregation Leads to Cancer: Lessons from BubR1 Mouse Models

Lee, Hyunsook Korean Society for Molecular and Cellular Biology 2014 Molecules and cells Vol.37 No.10

Alteration in chromosome numbers and structures instigate and foster massive genetic instability. As Boveri has seen a hundred years ago (Boveri, 1914; 2008), aneuploidy is hall-mark of many cancers. However, whether aneuploidy is the cause or the result of cancer is still at debate. The molecular mechanism behind aneuploidy includes the chromosome mis-segregation in mitosis by the compromise of spindle assembly checkpoint (SAC). SAC is an elaborate network of proteins, which monitor that all chromosomes are bipolarly attached with the spindles. Therefore, the weakening of the SAC is the major reason for chromosome number instability, while complete compromise of SAC results in detrimental death, exemplified in natural abortion in embryonic stage. Here, I will review on the recent progress on the understanding of chromosome missegregation and cancer, based on the comparison of different mouse models of BubR1, the core component of SAC.

Upregulation of Dendritic Arborization by N-acetyl-D-Glucosamine Kinase Is Not Dependent on Its Kinase Activity

Lee, HyunSook,Dutta, Samikshan,Moon, Il Soo Korean Society for Molecular and Cellular Biology 2014 Molecules and cells Vol.37 No.4

N-acetylglucosamine kinase (GlcNAc kinase or NAGK; EC 2.7.1.59) is highly expressed and plays a critical role in the development of dendrites in brain neurons. In this study, the authors conducted structure-function analysis to verify the previously proposed 3D model structure of GlcNAc/ATP-bound NAGK. Three point NAGK mutants with different substrate binding capacities and reaction velocities were produced. Wild-type (WT) NAGK showed strong substrate preference for GlcNAc. Conversion of Cys143, which does not make direct hydrogen bonds with GlcNAc, to Ser (i.e., C143S) had the least affect on the enzymatic activity of NAGK. Conversion of Asn36, which plays a role in domain closure by making a hydrogen bond with GlcNAc, to Ala (i.e., N36A) mildly reduced NAGK enzyme activity. Conversion of Asp107, which makes hydrogen bonds with GlcNAc and would act as a proton acceptor during nucleophilic attack on the ${\gamma}$-phosphate of ATP, to Ala (i.e., D107A), caused a total loss in enzyme activity. The overexpression of EGFP-tagged WT or any of the mutant NAGKs in rat hippocampal neurons (DIV 5-9) increased dendritic architectural complexity. Finally, the overexpression of the small, but not of the large, domain of NAGK resulted in dendrite degeneration. Our data show the effect of structure on the functional aspects of NAGK, and in particular, that the small domain of NAGK, and not its NAGK kinase activity, plays a critical role in the upregulation of dendritogenesis.

Characterization of three point mutations at the GlcNAc binding pocket of murine GlcNAc kinase and their effects on dendritic arborization

HyunSook Lee,Ji Hye Lee,Min-Jin Kang,Md. Abdul Hannan,Il Soo Moon 한국당과학회 2012 한국당과학회 학술대회 Vol.2012 No.1

N-acetylglucosamine (GlcNAc) kinase (NAGK; EC 2.7.1.59) converts GlcNAc into GlcNAc-6-phosphate. Based on the proposed 3D structure, we produced 3 point mutant NAGKs that were expected to retain differential capacities for substrate binding and reaction velocity. The proteins were expressed in Escherichia coli, affinity-purified to homogeneity, and used for functional analysis. Among the mutants, conversion of Cys143, which does not make direct hydrogen bonds with GlcNAc to Ser (i.e., C143S) had the least affecton enzymatic activity. Conversion of Asn36, which plays a role in domain closure by making a hydrogen bond with GlcNAc to Ala (i.e., N36A) mildly reduced the enzyme activity. Conversion of Asp107, which makes hydrogen bonds with GlcNAc and acts as a proton acceptor to Ala (i.e., D107A), caused a total loss in the enzyme activity. The eGFP- or RFP (DsRed)-tagged mutant NAGKs increased the complexity of dendritic architecture when overexpressed in rat hippocampal neurons (DIV 5-9) with no statistical difference with wild-type NAGK. These results indicate that the upregulation of dendritic complex by NAGK is the enzyme’s non-canonical function.

Advanced tube formation assay using human endothelial colony forming cells for in vitro evaluation of angiogenesis

Lee, Hyunsook,Kang, Kyu-Tae The Korean Society of Pharmacology 2018 The Korean Journal of Physiology & Pharmacology Vol.22 No.6

The tube formation assay is a widely used in vitro experiment model to evaluate angiogenic properties by measuring the formation of tubular structures from vascular endothelial cells (ECs). In vitro experimental results are crucial when considered the advisability of moving forward to in vivo studies. Thus, the additional attentions to the in vitro assay is necessary to improve the quality of the pre-clinical data, leading to better decision-making for successful drug discovery. In this study, we improved the tube formation assay system in three aspects. First, we used human endothelial colony forming cells (ECFCs), which are endothelial precursors that have a robust proliferative capacity and more defined angiogenic characteristics compared to mature ECs. Second, we utilized a real-time cell recorder to track the progression of tube formation for 48 hours. Third, to minimize analysis error due to the limited observation area, we used image-stitching software to increase the microscope field of view to a $2{\times}2$ stitched area from the $4{\times}$ object lens. Our advanced tube formation assay system successfully demonstrated the time-dependent dynamic progression of tube formation in the presence and absence of VEGF and FGF-2. Vatalanib, VEGF inhibitor, was tested by our assay system. Of note, $IC_{50}$ values of vatalanib was different at each observation time point. Collectively, these results indicate that our advanced tube formation assay system replicates the dynamic progression of tube formation in response to angiogenic modulators. Therefore, this new system provides a sensitive and versatile assay model for evaluating pro- or anti-angiogenic drugs.

Prognostic value of alternative lengthening of telomeres-associated biomarkers in uterine sarcoma and uterine carcinosarcoma.

Lee, Yoo-Kyung,Park, Noh-Hyun,Lee, Hyunsook Blackwell Scientific Publications 2012 International journal of gynecological cancer Vol.22 No.3

<P>A subset of cancer cells maintains telomere lengths in a telomerase-independent manner known as the alternative lengthening of telomeres (ALT). The goal of this study was to evaluate the frequency of ALT in uterine sarcoma and carcinosarcoma and to assess its association with clinical parameters.</P>

Clinicopathological values of NBS1 and DNA damage response genes in epithelial ovarian cancers

Lee, Yoo-Kyung,Park, Noh-Hyun,Lee, Hyunsook Nature Publishing Group 2015 Experimental and molecular medicine Vol.47 No.11

<P>Epithelial ovarian cancers (EOCs) are highly lethal gynecological malignancies with a high recurrence rate. Therefore, developing prognostic markers for recurrence after chemotherapy is crucial for the treatment of ovarian cancers. As ovarian cancers frequently respond to DNA-damaging agents, we assessed the clinicopathological significance of key double-strand DNA break (DSB) repair genes, including <I>BRCA1</I>, <I>BRCA2</I>, <I>BARD1</I>, <I>ATM</I>, <I>RAD51</I> and <I>NBS1</I> in EOC cell lines and paraffin-embedded tissue sections from 140 EOC patients treated with cytoreductive surgery, followed by platinum-based chemotherapy. These samples were analyzed for the clinicopathological impact of DSB genes by western blot analysis, immunohistochemistry and quantitative real-time PCR. Of the DSB repair genes, <I>BRCA1</I>, <I>ATM</I> and <I>NBS1</I>, which are involved in the homologous recombination-mediated repair pathway, were related to aggressive parameters in EOC. When survival analysis was performed, <I>NBS1</I> expression exhibited an association with EOC recurrence. Specifically, increased <I>NBS1</I> expression was found in 107 out of 140 cases (76.0%) and correlated with advanced stage (<I>P</I>=0.001), high grade (<I>P</I>=0.001) and serous histology (<I>P</I>=0.008). The median recurrence-free survival in patients with positive and negative expression of <I>NBS1</I> was 30 and 78 months, respectively (<I>P</I>=0.0068). In multivariate analysis, NBS1 was an independent prognostic factor for the recurrence of EOC. Together, these results suggest that NBS1 is a marker of poor prognosis for the recurrence of EOC and is associated with aggressive clinicopathological parameters.</P>

Nonthermal Broadening of UV lines Observed at the Limb of the Quiet Sun

Hyunsook Lee,Hong Sik Yun,Jongchul Chae 한국천문학회 1999 天文學會報 Vol.24 No.2

Vasculogenic property of endothelial colony forming cells combined with mesenchymal progenitor cells in high glucose condition.

LEE Hyunsook,KANG Kyu-Tae 한국특수교육학회 2014 한국특수교육학회 학술대회 Vol.2014 No.-