• Proceedings of the Korean Society of Sericultural Science Conference
• /
• 2003.10a
• /
• pp.97-98
• /
• 2003

Current study was aimed to understand an interaction between Dopa decarboxylase (DDC) and proteins that specifically binds to DDC in the silkworm, Bombyx mori. Materials and Methods: Materials-Animal: Bombyx mori Construction of GST fusion protein Preparation of lysates: Protein extracted from whole body of Bombyx mori Methods-In vitro binding assay with lysates, Peptide sequence and RACE-PCR (omitted)

• Journal of Microbiology and Biotechnology
• /
• v.5 no.3
• /
• pp.132-137
• /
• 1995

The lambda integrase (lnt) is believed to bind to several arm and core sites of attP DNA in order to facilitate intasome formation. We have done systematic mutagenic analysis on all 5 arm sites and found that P1 is absolutely required for integration while P2 is not. We also found that all 3 P' arm sites(P'1, P'2, and P'3) are required for efficient integrative recombination. P'1, which is an important binding site for excision, also seems to be crucial for integration when preincubation of attP DNA with Int and IHF is performed before recombination. Preincubation assay revealed that preincubation with Int and IHF improved the efficiency of recombination of wild type attP DNA and demolished recombinations of P'1 mutant attP DNAs.

• Journal of Gastric Cancer
• /
• v.22 no.4
• /
• pp.348-368
• /
• 2022

Purpose: Chromosomal instability is a hallmark of gastric cancer (GC). It can be driven by single nucleotide variants (SNVs) in cell cycle genes. We investigated the associations between SNVs in candidate genes, PLK2, PLK3, and ATM, and GC risk and clinicopathological features. Materials and Methods: The genotyping study included 542 patients with GC and healthy controls. Generalized linear models were used for the risk and clinicopathological association analyses. Survival analysis was performed using the Kaplan-Meier method. The binding of candidate miRs was analyzed using a luciferase reporter assay. Results: The PLK2 C_rs15009-C_rs963615 haplotype was under-represented in the GC group compared to that in the control group (P_corr=0.050). Male patients with the PLK2 rs963615 CT genotype had a lower risk of GC, whereas female patients had a higher risk (P=0.023; P=0.026). The PLK2 rs963615 CT genotype was associated with the absence of vascular invasion (P=0.012). The PLK3 rs12404160 AA genotype was associated with a higher risk of GC in the male population (P=0.015). The ATM T_rs228589-A_rs189037-G_rs4585 haplotype was associated with a higher risk of GC (P<0.001). The ATM rs228589, rs189037, and rs4585 genotypes TA+AA, AG+GG, and TG+GG were associated with the absence of perineural invasion (P=0.034). In vitro analysis showed that the cancer-associated miR-23b-5p mimic specifically bound to the PLK2 rs15009 G allele (P=0.0097). Moreover, low miR-23b expression predicted longer 10-year survival (P=0.0066) in patients with GC. Conclusions: PLK2, PLK3, and ATM SNVs could potentially be helpful for the prediction of GC risk and clinicopathological features. PLK2 rs15009 affects the binding of miR-23b-5p. MiR-23b-5p expression status could serve as a prognostic marker for survival in patients with GC.

• Korean Journal of Animal Reproduction
• /
• v.18 no.2
• /
• pp.95-99
• /
• 1994

The purpose of the present study was to determine the in vivo effect of oxytocin antagonist-I(Al) on uterine oxytocin receptor number (Rn) and/or binding affinity (Kd) in the estrous rat. Anesthetized rats were given a bolus infusion of control or 5${\mu}\textrm{g}$ of AI and sacri-ficed 0.5 and 4 hours later. The uterine tissue was removed, trimmed and frozen. Membrane oxytocin receptors were isolated after homogenization of uterine tissue and differential ultracentrifugation. The oxytocin receptor assay was performed by saturation with cold oxytocin competion with a high specific activity oxytocin antagonist. Rn and Kds were determined by nonlinear curve fitting methods. No differences(p>0.05) between the AI and control treated animals in either oxytocin receptor number or binding affinity was detected in this study. These data suggest that the major mode of action of AI is via competitive inhibition at the uterine oxytocin receptor and not by altering receptor number or binding affinity.

• Journal of Ginseng Research
• /
• v.48 no.2
• /
• pp.171-180
• /
• 2024

Background: Epimers of ginsenoside Rg3 (Rg3) have a low bioavailability and are prone to deglycosylation, which produces epimers of ginsenoside Rh2 (S-Rh2 and R-Rh2) and protopanaxadiol (S-PPD and R-PPD). The aim of this study was to compare the efficacy and potency of these molecules as anti-cancer agents. Methods: Crystal violet staining was used to study the anti-proliferatory action of the molecules on a human epithelial breast cancer cell line, MDA-MB-231, and human umbilical vein endothelial cells (HUVEC) and compare their potency. Cell death and cell cycle were studied using flow cytometry and mode of cell death was studied using live cell imaging. Anti-angiogenic effects of the drug were studied using loop formation assay. Molecular docking showed the interaction of these molecules with vascular endothelial growth factor receptor-2 (VEGFR2) and aquaporin (AQP) water channels. VEGF bioassay was used to study the interaction of Rh2 with VEGFR2, in vitro. Results: HUVEC was the more sensitive cell line to the anti-proliferative effects of S-Rh2, S-PPD and R-PPD. The molecules induced necroptosis/necrosis in MDA-MB-231 and apoptosis in HUVEC. S-Rh2 was the most potent inhibitor of loop formation. In silico molecular docking predicted a good binding score between Rh2 or PPD and the ATP-binding pocket of VEGFR2. VEGF bioassay showed that Rh2 was an allosteric modulator of VEGFR2. In addition, SRh2 and PPD had good binding scores with AQP1 and AQP5, both of which play roles in cell migration and proliferation. Conclusion: The combination of these molecules might be responsible for the anti-cancer effects observed by Rg3.

• Journal of Microbiology and Biotechnology
• /
• v.12 no.4
• /
• pp.592-597
• /
• 2002

The Rkp1/CPC2, a receptor for activated protein kinase C of Schizosaccharomyces pombe, contains seven WD motifs found in the G-protein $\beta$-subunit. A 110-kDa protein was identified to interact with Rkp1/CPC2 by immunoprecipitation and following in vitro binding assay. To examine its kinase activity and binding ability to Rkp1, the $pck1^+$, a PKC homolog of S. pombe, was cloned. Pckl phosphorylated myelin basic protein (MBP) and histone Hl in a phospholipid-dependent and $Ca^{2+}$-independent manner. It was demonstrated that the N-terminal region of Pck1 was responsible for the binding to Rkp1 , thus suggesting that Rkp1 interacted with Pckl to regulate Pckl-mediated signaling in S. pombe.

• BMB Reports
• /
• v.36 no.2
• /
• pp.207-213
• /
• 2003

Peroxisome proliferator-activated receptors (PPARs) are orphan nuclear hormone receptors that are known to control the expression of genes that are involved in lipid homeostasis and energy balance. PPARs activate gene transcription in response to a variety of compounds, including hypolipidemic drugs. Most of these compounds have high affinity to the ligand-binding domain (LBD) of PPARs and cause a conformational change within PPARs. As a result, the receptor is converted to an activated mode that promotes the recruitment fo co-activators such as the steroid receptor co-activator-1 (SRC-1). Based on the activation mechanism of PPARs (the ligand binding to $PPAR{\gamma}$ induces interactions of the receptor with transcriptional co-activators), we performed Western blot and ELISA. These showed that the indomethacin, a $PPAR{\gamma}$ ligand, increased the binding between $PPAR{\gamma}$ and SRC-1 in a ligand dose-dependent manner. These results suggested that the in vitro conformational change of $PPAR{\gamma}$ by ligands was also induced, and increased the levels of the ligand-dependent interaction with SRC-1. Collectively, we developed a novel and useful ELISA system for the mass screening of $PPAR{\gamma}$ ligands. This screening system (based on the interaction between $PPAR{\gamma}$ and SRC-1) may be a promising system in the development of drugs for metabolic disorders.

• Biomolecules & Therapeutics
• /
• v.27 no.2
• /
• pp.201-209
• /
• 2019

Mixed lineage leukemia proteins (MLL) are the key histone lysine methyltransferases that regulate expression of diverse genes. Aberrant activation of MLL promotes leukemia as well as solid tumors in humans, highlighting the urgent need for the development of an MLL inhibitor. We screened and isolated MLL1-binding ssRNAs using SELEX (${\underline{S}}ystemic$ ${\underline{E}}volution$ of ${\underline{L}}igands$ by ${\underline{E}}xponential$ enrichment) technology. When sequences in sub-libraries were obtained using next-generation sequencing (NGS), the most enriched aptamers-APT1 and APT2-represented about 30% and 26% of sub-library populations, respectively. Motif analysis of the top 50 sequences provided a highly conserved sequence: 5'-A[A/C][C/G][G/U][U/A]ACAGAGGG[U/A]GG[A/C] GAGUGGGU-3'. APT1, APT2, and APT5 embracing this motif generated secondary structures with similar topological characteristics. We found that APT1 and APT2 have a good binding activity and the analysis using mutated aptamer variants showed that the site information in the central region was critical for binding. In vitro enzyme activity assay showed that APT1 and APT2 had MLL1 inhibitory activity. Three-dimensional structure prediction of APT1-MLL1 complex indicates multiple weak interactions formed between MLL1 SET domain and APT1. Our study confirmed that NGS-assisted SELEX is an efficient tool for aptamer screening and that aptamers could be useful in diagnosis and treatment of MLL1-mediated diseases.

• Asian-Australasian Journal of Animal Sciences
• /
• v.18 no.12
• /
• pp.1708-1714
• /
• 2005

Keratinocyte growth factor (KGF) functions in epithelial growth and differentiation in many tissues and organs. KGF is expressed in the uterine endometrial epithelial cells during the estrous cycle and pregnancy in pigs, and receptors for KGF (KGFR) are expressed by conceptus trophectoderm and endometrial epithelia. KGF has been shown to stimulate the proliferation and differentiation of conceptus trophectoderm. However, the role of KGF on the endometrial epithelial cells has not been determined. Therefore, this study determined the effect of KGF on proliferation and differentiation of endometrial epithelial cells in vitro and in vivo using an immortalized porcine luminal epithelial (pLE) cell line and KGF infusion into the uterine lumen of pigs between Days 9 and 12 of estrous cycle. Results showed that KGF did not stimulate proliferation of uterine endometrial epithelial cells in vitro and in vivo determined by the $^3$H]thymidine incorporation assay and the proliferating cell nuclear antigen staining, respectively. Effects of KGF on expression of several markers for epithelial cell differentiation, including integrin receptor subunits $\alpha$4, $\alpha$5 and $\beta$1, plasmin/trypsin inhibitor, uteroferrin and retinol-binding protein were determined by RT-PCR, Northern and slot blot analyses, and immunohistochemisty, and KGF did not affect epithelial cell differentiation in vitro and in vivo. These results show that KGF does not induce epithelial cell proliferation and differentiation, suggesting that KGF produced by endometrial epithelial cells acts on conceptus trophectoderm in a paracrine manner rather than on endometrial epithelial cells in an autocrine manner.

• Animal cells and systems
• /
• v.7 no.3
• /
• pp.227-235
• /
• 2003

Bisphenol A (BPA), nonylphenol (NP), and 4-tert-octylphenol (OP) are known endocrine disrupting chemicals (EDCs) with estrogenic activity in fish. This study compared the effects of BPA, NP and OP on in vitro vitellogenin (VTG) synthesis in primary cultures of hepatocytes of the Chinese minnow Phoxinus oxycephalus. The VTG secreted into the culture medium was measured using enzyme-linked immunosorbent assay (ELISA), which we developed in this study using an antibody prepared from homogenates of Chinese minnow egg. VTG synthesis was induced by estradiol-17$\beta$ ($E_2$) and phenols (BPA, NP and OP) treatment. $E_2$ at concentrations of 10$^{-6}$ M or higher increased VTG levels significantly (P < 0.05). Exposure to 10^5\;M\;BPA\;or\;10^-4$M NP and OPinduced in vitro VTG synthesis (P < 0.01). However, $10^-3$ M BPA, NP or OP did not induce VTG synthesis. These results suggest that SPA has the highest estrogenic potential in Chinese minnow hepatocytes. Tamoxifen, an anti-estrogen, drastically blocked the production of VTG by phenols (BPA, NP and OP) suggesting that phenols (BPA, NP and OP) may act via binding to estrogen receptor (ER) in Chinese minnow hepatocytes.