• Archives of Pharmacal Research
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• v.27 no.9
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• pp.961-967
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• 2004

The development of an antibody labeling method with $^{99m}$Tc is important for cancer imaging. Most bifunctional chelate methods for $^{99m}$Tc labeling of antibody incorporate a $^{99m}$Tc chelator through a linkage to lysine residue. In the present study, a novel site-specific $^{99m}$Tc labeling method at carbohydrate side chain in the Fc region of 2 antibodies (T101 and rabbit anti-human serum albumin antibody (RPAb)) using dihydrazinophthalazine (DHZ) which has 2 hydrazino groups was developed. The antibodies were oxidized with sodium periodate to pro-duce aldehyde on the Fc region. Then, one hydrazine group of DHZ was conjugated with an aldehyde group of antibody through the formation of a hydrazone. The other hydrazine group was used for labeling with $^{99m}$Tc. The number of conjugated DHZ was 1.7 per antibody. $^{99m}$Tc labeling efficiency was 46-85％ for T101 and 67∼87％ for RPAb. Indirect labeling with DHZ conjugated antibodies showed higher stability than direct labeling with reduced antibodies. High immunoreactivities were conserved for both indirectly and directly labeled antibodies. A biodistribution study found high blood activity related to directly labeled T1 01 at early time point as well as low liver activity due to indirectly labeled T101 at later time point. However, these findings do not affect practical use. No significantly different biodistribution was observed in the other organs. The research concluded that DHZ can be used as a site-specific bifunctional chelating agent for labeling antibody with $^{99m}$Tc. Moreover, $^{99m}$Tc labeled antibody via DHZ was found to have excellent chemical and biological properties for nuclear medicine imaging.edicine imaging.

• Journal of Ginseng Research
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• v.42 no.2
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• pp.165-174
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• 2018

Background: Extended endoplasmic reticulum (ER) stress may initiate apoptotic pathways in cancer cells, and ER stress has been reported to possibly increase tumor death in cancer therapy. We previously reported that caspase-8 played an important role in compound K-induced apoptosis via activation of caspase-3 directly or indirectly through Bid cleavage, cytochrome c release, and caspase-9 activation in HL-60 human leukemia cells. The mechanisms leading to apoptosis in A549 and SK-MES-1 human lung cancer cells and the role of ER stress have not yet been understood. Methods: The apoptotic effects of compound K were analyzed using flow cytometry, and the changes in protein levels were determined using Western blot analysis. The intracellular calcium levels were monitored by staining with Fura-2/AM and Fluo-3/AM. Results: Compound K-induced ER stress was confirmed through increased phosphorylation of $eIF2{\alpha}$ and protein levels of GRP78/BiP, XBP-1S, and $IRE1{\alpha}$ in human lung cancer cells. Moreover, compound-K led to the accumulation of intracellular calcium and an increase in m-calpain activities that were both significantly inhibited by pretreatment either with BAPTA-AM (an intracellular $Ca^{2+}$ chelator) or dantrolene (an RyR channel antagonist). These results were correlated with the outcome that compound K induced ER stress-related apoptosis through caspase-12, as z-ATAD-fmk (a specific inhibitor of caspase-12) partially ameliorated this effect. Interestingly, 4-PBA (ER stress inhibitor) dramatically improved the compound K-induced apoptosis. Conclusion: Cell survival and intracellular $Ca^{2+}$ homeostasis during ER stress in human lung cancer cells are important factors in the induction of the compound K-induced apoptotic pathway.

• Journal of Radiopharmaceuticals and Molecular Probes
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• v.2 no.2
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• pp.108-112
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• 2016

Molecular imaging with the radiolabeled RGD peptides for ${\alpha}_v{\beta}_3$ integrin has been an increasing interest for tumor diagnosis and the treatment monitoring. Recently, $^{64}Cu$-NODAGA-gluco-E[c(RGDfK)]$_2$ was developed for quantification of ${\alpha}_v{\beta}_3$ integrin and its biological properties was elucidated. To better understand the molecular process in vivo, we performed the kinetic analysis for the $^{64}Cu$-NODAGA-gluco-E[c(RGDfK)]$_2$. After preparation of a radiotracer, dynamic PET images were obtained in the U87MG xenograft mice for 60 min (n = 6). Binding potential values were estimated from the 3-tissue compartment model, reference Logan and simplified reference tissue model. In the early time frame (0-20 min), the liver, kidney, intestine, urinary bladder and tumor were visualized but these uptakes were diminished as time went by. The tumors showed a good contrast at 40 min after administration. $^{64}Cu$-NODAGA-gluco-E[c(RGDfK)]$_2$ showed the 2-fold uptake in the tumor compared with that in the muscle. The parametric maps for binding values also provide the higher tumor-to-background contrast than the static images. A binding value obtained from the 3-tissue compartment model was comparable to other modeling methods. From these results, we conclude that $^{64}Cu$-NODAGA-gluco-E[c(RGDfK)]$_2$ may be a promising PET radiotracer for the evaluation of angiogenesis.

• Journal of Radiopharmaceuticals and Molecular Probes
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• v.2 no.2
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• pp.118-122
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• 2016

Integrin ${\alpha}_v{\beta}_3$ plays an important role in the tumor metastases and angiogenesis. Arginine-glycine-aspartate (RGD) peptide motif binds to the integrin ${\alpha}_v{\beta}_3$. General $^{68}Ga$-labeled cyclic RGD peptides was rapidly eliminated from the circulatory system by renal excretion because of its hydrophilic property. The purpose of this study was to develop a novel $^{68}Ga$-labeled cyclic RGD peptides, which could acquire enhanced PET tumor images with improved pharmacokinetics by adopting biphenyl group between chelator and RGD peptides. $^{68}Ga$-DOTA-2P-c(RGDyK) was demonstrated a 12% higher lipophilicity level than $^{68}Ga$-DOTA-c(RGDyK) as a reference compound. In the animal PET, $^{68}Ga$-DOTA-2P-c(RGDyK) represented relatively lower blood-clearance, and an increased signal to noise ratio compared to $^{68}Ga$-DOTA-c(RGDyK). From these perspective, $^{68}Ga$-DOTA-2P-c(RGDyK) could be a good candidate for in integrin ${\alpha}_v{\beta}_3$-expressed tumor imaging.

• Journal of Radiation Protection and Research
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• v.19 no.3
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• pp.222-229
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• 1994

Radiostrontium passes the placental barrier in pregnant rodents very well. Chitosan, a natural nontoxic chelator, was reported to reduce whole body retention of radiostrontium in mice. The aim of the present study was to evaluate water soluble chitosan as a blocking agent of transplacental transfer of radiostrontium in pregnant mice. Twenty pregnant mice were divided into four groups: control and three groups of chitosan treatment (groups 1 to 3). Sr-85(15KBq in 0.2ml saline) was subcutaneously injected into pregnant mice at the 17th day of pregnancy. In control mice, 0.2ml saline was given 5 hours after the injection of Sr-85. In group 1, 1% water soluble chitosan was given subcutanously for two days, twice daily after the injection of Sr-85. In group 2, 10% water soluble chitosan was given orally for 15 days before conception. In group 3, 0.3% water soluble chitosan was injected intravenously for 15 days, once daily before conception. Gamma counting of newborns were done at days 0, 2 and 7 after their births. Whole body retention of Sr-85 in newborns of control mice at days 0, 2, 7 were $3.1{\pm}0.3%,\;2.9{\pm}0.3%,\;2.8{\pm}0.3%$ respectively. In experimental groups, whole body retention of Sr-85 was significantly lower than that of control (p<0.01) and no statistical difference was noted between them. In group 1, the values were $2.1{\pm}0.3%,\;1.4{\pm}0.1%,\;1.4{\pm}0.1%$, respectively. In group 3, they were $2.1{\pm}0.2%,\;1.7{\pm}0.2%,\;1.6{\pm}0.2%$, respectively. In conclusion, the water soluble chitosan reduced transplacental contamination of radiostrontium in pregnant mice.

• Proceedings of the Korean Society of Crop Science Conference
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• 2017.06a
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• pp.36-36
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• 2017

Out of twenty common protein amino acids, there are many kinds of non protein amino acids (NPAAs) that exist as secondary metabolites and exert ecological functions in plants. Mimosine (Mim), one of those NPAAs derived from L. leucocephala acts as an iron chelator and reversely block mammalian cell cycle at G1/S phases. Cysteine (Cys) is decisive for protein and glutathione that acts as an indispensable sulfur grantor for methionine and many other sulfur-containing secondary products. Cys biosynthesis includes consecutive two steps using two enzymes-serine acetyl transferase (SAT) and O-acetylserine (thiol)lyase (OASTL) and appeared in plant cytosol, chloroplast, and mitochondria. In the first step, the acetylation of the ${\beta}$-hydroxyl of L-serine by acetyl-CoA in the existence of SAT and finally, OASTL triggers ${\alpha}$, ${\beta}$-elimination of acetate from OAS and bind $H_2S$ to catalyze the synthesis of Cys. Mimosine synthase, one of the isozymes of the OASTLs, is able to synthesize Mim with 3-hydroxy-4-pyridone (3H4P) instead of $H_2S$ for Cys in the last step. Thus, the aim of this study was to clone and characterize the cytosolic (Cy) OASTL gene from L. leucocephala, express the recombinant OASTL in Escherichia coli, purify it, do enzyme kinetic analysis, perform docking dynamics simulation analysis between the receptor and the ligands and compare its performance between Cys and Mim synthesis. Cy-OASTL was obtained through both directional degenerate primers corresponding to conserved amino acid region among plant Cys synthase family and the purified protein was 34.3KDa. After cleaving the GST-tag, Cy-OASTL was observed to form mimosine with 3H4P and OAS. The optimum Cys and Mim reaction pH and temperature were 7.5 and $40^{\circ}C$, and 8.0 and $35^{\circ}C$ respectively. Michaelis constant (Km) values of OAS from Cys were higher than the OAS from Mim. Inter fragment interaction energy (IFIE) of substrate OAS-Cy-OASTL complex model showed that Lys, Thr81, Thr77 and Gln150 demonstrated higher attraction force for Cys but 3H4P-mimosine synthase-OAS intermediate complex showed that Gly230, Tyr227, Ala231, Gly228 and Gly232 might provide higher attraction energy for the Mim. It may be concluded that Cy-OASTL demonstrates a dual role in biosynthesis both Cys and Mim and extending the knowledge on the biochemical regulatory mechanism of mimosine and cysteine.

• Applied Microscopy
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• v.38 no.1
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• pp.1-10
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• 2008

Dandelion has been frequently used as a remedy for women's disease, inflammatory diseases and disorders of the liver and gallbladder. Dandelion extracts water extract, an herbal medication, may have an effect on the activity of hepatic antioxidant enzymes in diabetic rat. This study aims demonstrate the effect of dandelion extracts, one of the natural chelator, on the biochemical and enzyme activity changes in the mouse liver caused by $HgCl_2$. Mice approximately 30 gm in weight were grouped into the control, mercury chloride-treated, and the dandelion extracts-treated after mercury chloride groups. $HgCl_2$ (5 mg/kg) and dandelion extracts (3 g/kg) were delivered orally. Serum AST and ALT were measured, enzyme activity of liver were examined by spectrophotometer and ultrastructural alteration of liver were examined by light and electron microscopy. Dandelion extracts were decreased the increase of serum AST and ALT level induced by mercury. The catalase activity was decreased in the dandelion extracts group. The activity of SOD was dereased, but did not show significant differences. Mercury chloride-treated hepatic cell were irregular nucleus, enlarged and reduced number of mitochodria, enlarged rough endoplasmic reticulum, loss of ribosomes. Cells treated with dandelion extracts were similar to those of the control group. In conclusion, dandelion extracts may protect the mercury-induced toxicity on Liver.

• The Journal of Internal Korean Medicine
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• v.21 no.1
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• pp.13-22
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• 2000

목적 : 본(本) 연구(硏究)는 배기음(排氣飮)이 인간(人間)의 장관내(腸管內)에서 산화물(酸化物)에 의해 유발(誘發)된 세포(細胞)의 사망(死亡) 및 DNA의 손상(損傷)을 방지할수 있는지를 검증(檢證)하기 위한 실험(實驗)이다. 방법 : 배양(培養)된 인체장관(人體腸管) 세포계열(細胞系列)인 Caco-2 세포(細胞)에서 세포(細胞)의 사망(死亡)은 trypan bile의 소실정도에 의해서 평가했으며, DNA의 손상(損傷)은 double stranded DNA의 파괴정도를 측정하여 평가하였다. $H_2O_2$는 표본(標本) 산화제(酸化劑)로 사용되었다. 결과 : $H_2O_2$에 노출된 세포들의 세포사망(細胞死亡) 정도는 노출시간과 용량에 비례하여 증가하는 양상을 보였다. 배기음(排氣飮)은 $H_2O_2$에 의해 유발(誘發)되는 세포방지를 방지하였고, 0.05-1%의 농도범위에 걸쳐서는 그 효과가 용량에 비례하여 증가하는 양상을 보였다. $H_2O_2$에 의해 유발(誘發)된 세포손상(細胞損傷)은 catalase(hydrogen peroxide scavenger enzyme)와 deferoxamine(iron chelator)에 의해 억제되었다. 그러나 강력한 항산화제(抗酸化劑)인 DPPD는 $H_2O_2$에 의해 유발(誘發)되는 세포손상(細胞損傷)에는 영향을 주지 못했다. $H_2O_2$에 의해 유발(誘發)된 지질(脂質)의 과산화(過酸化)는 배기음(排氣飮)과 DPPD에 의해 억제되었다. $H_2O_2$에 의해 유발(誘發)된 DNA의 손상(損傷)은 배기음(排氣飮)에 의해 방지되었으며 용량에 의존하는 양상을 보였다. $H_2O_2$에 의해 유발(誘發)된 DNA의 손상은 catalase와 deferoxamine에 의해 억제되었지만 DPPD는 억제시키지 못했다. 배기음(排氣飮)은 $H_2O_2$에 의해 유발(誘發)된 ATP의 소실을 회복시켰다. 이러한 실험결과 $H_2O_2$에 의해 유발(誘發)된 세포(細胞)의 손상(損傷)은 지질(脂質)의 과산화(過酸化)와는 다른 독립적인 기전에 의해 일어남을 나타낸다. 결론 : 이러한 결과들로 볼 때 Caco-2 세포(細胞)에서 배기음(排氣飮)이 항산화작용(亢酸化作用)보다는 다른 기전을 통하여 Caco-2 세포안에서 산화제(酸化劑)에 의해 유발(誘發)된 세포(細胞)의 사망(死亡)와 DNA의 손상(損傷)을 방지할 수 있다는 것을 가리킨다. 따라서 본 연구(硏究)는 배기음(排氣飮)이 반응성산소기(反應性酸素基)에 의해 매개된 인체(人體) 위장관질환(胃腸管疾患)의 치료(治療)에 사용할 수 있을 가능성(可能性)이 있음을 제시하고 있다.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.30 no.5
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• pp.809-815
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• 1997

A series of experiments were conducted to study effects of osmolality and $Ca^{2+}$ on sperm motility in marbled sole, Limanda yokohamae. Spermatozoa were immotile when the milt was mixed with solutions (electrolyte or non-electrolyte) of lower osmolality than the average seminal plasma osmolality (351 mOsm/kg), but became motile after mixing milt with an hyperosmotic solution. However, with the osmolality above 1,639 mOsm/kg spermatozoa ceased their movement. When the milt was suspended in the $Ca^{2+}$ free artificial seawater (ASW) containing ethylenglycoltriamine (EGTA), the percentage of motile spermatozoa decreased in proportion to the increase of EGTA. Most spermatozoa were quiescent in a medium containing 3 mM EGTA. The motility of spermatozoa prevented by 3 mM EGTA was recovered by the subsequent addition of $Ca^{2+}$ over the concentration of 0.25 mM. Effects of calmodulin antagonist, trifluoperazin (TFP) on spermatozoa were examined to elucidate the possible functions of calmodulin in sperm motility. TFP immobilized spermatozoa 5n ASW at the concentration of 0.5 M. These findings suggest that calcium is an effective external factor in the initiation process of sperm motility.

• The Korean Journal of Physiology and Pharmacology
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• v.20 no.1
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• pp.101-109
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• 2016

Reducing $[Mg^{2+}]_o$ to 0.1 mM can evoke repetitive $[Ca^{2+}]_i$ spikes and seizure activity, which induces neuronal cell death in a process called excitotoxicity. We examined the issue of whether cultured rat hippocampal neurons preconditioned by a brief exposure to 0.1 mM $[Mg^{2+}]_o$ are rendered resistant to excitotoxicity induced by a subsequent prolonged exposure and whether $Ca^{2+}$ spikes are involved in this process. Preconditioning by an exposure to 0.1 mM $[Mg^{2+}]_o$ for 5 min inhibited significantly subsequent 24 h exposure-induced cell death 24 h later (tolerance). Such tolerance was prevented by both the NMDA receptor antagonist D-AP5 and the L-type $Ca^{2+}$ channel antagonist nimodipine, which blocked 0.1 mM $[Mg^{2+}]_o$-induced $[Ca^{2+}]_i$ spikes. The AMPA receptor antagonist NBQX significantly inhibited both the tolerance and the $[Ca^{2+}]_i$ spikes. The intracellular $Ca^{2+}$ chelator BAPTA-AM significantly prevented the tolerance. The nonspecific PKC inhibitor staurosporin inhibited the tolerance without affecting the $[Ca^{2+}]_i$ spikes. While $G{\ddot{o}}6976$, a specific inhibitor of $PKC{\alpha}$ had no effect on the tolerance, both the $PKC{\varepsilon}$ translocation inhibitor and the $PKC{\zeta}$ pseudosubstrate inhibitor significantly inhibited the tolerance without affecting the $[Ca^{2+}]_i$ spikes. Furthermore, JAK-2 inhibitor AG490, MAPK kinase inhibitor PD98059, and CaMKII inhibitor KN-62 inhibited the tolerance, but PI-3 kinase inhibitor LY294,002 did not. The protein synthesis inhibitor cycloheximide significantly inhibited the tolerance. Collectively, these results suggest that low $[Mg^{2+}]_o$ preconditioning induced excitotoxic tolerance was directly or indirectly mediated through the $[Ca^{2+}]_i$ spike-induced activation of $PKC{\varepsilon}$ and $PKC{\xi}$, JAK-2, MAPK kinase, CaMKII and the de novo synthesis of proteins.