• The Journal of Korean Medicine
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• v.34 no.3
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• pp.126-142
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• 2013

Objectives: Previous reports have shown that Bogijetong-Tang (BJT) is effective in peripheral neuropathy induced by taxol and crush injury. In this study, we researched the effects of BJT on diabetic neuropathy induced by STZ in the mouse. Methods: We performed both in vitro and in vivo experiments to verify the effects of BJT on diabetic neuropathy induced by STZ in mice. Changes in axonal recovery were observed with immunofluorescence staining using NF-200, Hoechst33258, $S100{\beta}$, caspase 3 and anti-cdc2. Proliferation and degeneration of Schwann cells were investigated by immunofluorescence staining and western blot analyses. Results: BJT showed considerable effects on neurite outgrowth and axonal regeneration in diabetic neuropathy. BJT contributed to the creation of NF-200, GAP-43, Cdc2, phospho-vimentin, ${\beta}1$, active ${\beta}1$, ${\beta}3$ integrin, phospho-Erk1/2 protein. Conclusions: Through this study, we found that BJT is effective for enhanced axonal regeneration via dynamic regulation of regeneration-associated proteins. Therefore, BJT had a pharmaceutical property enhancing recovery of peripheral nerves induced by diabetic neuropathy and could be a candidate for drug development after more research.

• Journal of Embryo Transfer
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• v.18 no.3
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• pp.243-248
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• 2003

Sperrnatogenesis, the process by which the male germ-line stem cells(GSCs; type A spermatogonia) divide and differentiate to produce the mature spermatozoa, occurs in the seminiferous tubules of the testis. The GSCs proliferate actively to produce two types of cells: other GSCs and differentiating spermatogonia. GSCs have unipotentcy, devoted solely to the generation of sperm. The function of GSCs has broad implications for development, disease, and evolution. Spermatogenesis is fundamental for propagation of species and the defects of this system can result in infertility or disease. The ability to identify, isolate, culture, and alter GSCs will allow powerful new approaches in animal transgenesis and human gene therapy relating to infertility. Until recently, research on stem cells in the testis has been limited because of technical difficulties in isolating and identifying these cell populations. Here, we were trying to find out optimal conditions for in vitro culture of GSCs for identifying and isolating GSCs. We collected mouse GSCs from 3-days old mouse by two-step enzyme digestion method. GSCs were plated and grown on mouse embryonic fibroblasts in Dulbecco's modified Eagle's medium (DMEM) containing 15％ fatal bovine serum, 10 mM 2-mercaptoethanol, 1％ non-essential amino acids, 1 ng/$m\ell$ bFGF, 10 $\mu$M forskolin, 1500 U/$m\ell$ human recombinant leukemia inhibitory factor (LIF). Over a period 3∼5 days, GSCs gave rise to large multicellular colonies resembling those of mouse pluripotent stem cells. After 5th passages, cells within the colonies continued to be alkaline phosphatase and Oct-4 positive and tested positive against a panel of two immunological markers(Integrin $\alpha$ 6 and Integrin $\beta$ 1) that have been recognized generally to characterize GSCs. SSEA-1, SSEA-3, and SSEA-4 also showed positive signals. Based on our data, these GSCs-derived cultures meet the criteria for GSCs itself and even other pluripotent stem cells. We reported here the establishment of in vitro cultures from mouse male GSCs.

• IMMUNE NETWORK
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• v.23 no.3
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• pp.25.1-25.15
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• 2023

Mucosal environments harbour lymphocytes, which express several adhesion molecules, including intestinal homing receptors and integrin αE/β7 (CD103). CD103 binds E-cadherin, an integrin receptor expressed in intestinal endothelial cells. Its expression not only enables homing or retention of T lymphocytes at these sites but is also associated with increased T lymphocyte activation. However, it is not yet clear how CD103 expression is related to the clinical staging of breast cancer, which is determined by factors such as the size of the tumor (T), the involvement of nearby lymph nodes (N), and presence of metastasis (M). We examined the prognostic significance of CD103 by FACS in 53 breast cancer patients and 46 healthy controls enrolled, and investigated its expression, which contributes to lymphocyte recruitment in tumor tissue. Patients with breast cancer showed increased frequencies of CD103⁺, CD4⁺CD103⁺, and CD8⁺CD103⁺ cells compared to controls. CD103 was expressed at a high level on the surfaces of tumor-infiltrating lymphocytes in patients with breast cancer. Its expression in peripheral blood was not correlated with clinical TNM stage. To determine the localisation of CD103⁺ cells in breast tissue, tissue sections of breast tumors were stained for CD103. In tissue sections of breast tumors stained for CD103, its expression in T lymphocytes was higher compared to normal breast tissue. In addition, CD103⁺ cells expressed higher levels of receptors for inflammatory chemokines, compared to CD103^- cells. CD103⁺ cells in peripheral blood and tumor tissue might be an important source of tumor-infiltrating lymphocyte trafficking, homing, and retention in cancer patients.

• Journal of the Korean Magnetic Resonance Society
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• v.11 no.1
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• pp.10-23
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• 2007

A new disintegrin protein named saxatilin was purified from Korean snake venom (Gloydius saxatilis). Saxatilin is a 73 residue small ploypeptide, which has a primary recognition motif in extracellular matrix, Arg-Gly-Asp (RGD) sequence. Data from inhibition activity assay for the ${\alpha}_v{\beta}_3$ integrin showed that saxatilin showed about 5000-fold higher activity than those of RGD peptides, suggesting that RGD sequence may not be sufficient to induce full cellular function of this site. The solution structures calculated from NMR data were well converged for backbone atoms except RGD loop. The structure revealed that most of tight turns are stabilized by medium range NOE contacts and the RGD motif is located far from the rigid core of the C-terminal domain. The three-dimensional fold and biological function of saxatilin are discussed with those of salmosin, which is a disintegrin protein derived from Agkistrodon halys brevicaudus.

• International Journal of Oral Biology
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• v.32 no.1
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• pp.35-43
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• 2007

Tooth loss in elderly is mainly caused by alveolar bone loss via severe periodontitis. Although the severity of periodontitis is known to be affected by age, the aging process or the genetic changes during the aging of periodontal tissue cells are not well characterized. In this study, we investigated the effect of in vitro aging on the change of gene expression pattern in periodontal fibroblasts. Gingival fibroblasts (GF) and periodontal ligament fibroblasts (PDL) were obtained from two young patients and replicative senescence was induced by sequential subcultivation. When more than 90% cells were positively stained with senescence-associated ${\beta},-galactosidase$, those cells were regarded as aged cells. In aged GF and PDL, the level of phosphorylated retinoblastoma (RB) and $p16^{INK4A}$ protein was significantly decreased and increased, respectively. However, the protein level of p53 and p21, well known senescence-inducing genes, did not increase in aged GF and PDL. Although $p27^{Kip1}$ and $p15^{INK4B}$, another cyclin-dependent kinase inhibitors, were reported to be involved in replicative senescence of human cells, they were decreased in aged GF and PDL. Because senescent cells showed flattened and enlarged cell shape and are known to have increased focal adhesion, we examined the protein level of several integrins. Aged GF and PDL showed increased protein level of integrin ${\alpha}2$, ${\alpha}v$, and ${\beta}1$. When the gene expression profiles of actively proliferating young cells and aged cells were compared by cDNA microarray of 3,063 genes and were confirmed by reverse transcription-polymerase chain reaction, 7 genes and 15 genes were significantly and commonly increased and decreased, respectively, in aged GF and PDL. Among them, included are the genes that were known to be involved in the regulation of cell cycle, gene transcription, or integrin signaling. The change of gene expression pattern in GF and PDL was minimally similar to that of oral keratinocyte. These results suggest that $p16^{INK4A}/RB$ might be involved in replicative senescence of periodontal fibroblasts and the change of gene expression profile during aging process is cell type specific.

• Animal cells and systems
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• v.5 no.3
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• pp.253-262
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• 2001

Ligand-receptor clustering event is the most important step in leukocyte adhesion and spreading on endothelial cells. Intercellular adhesion molecule-1 (ICAM-1) has been shown to enhance leukocyte adhesion, but the molecular event during the process of adhesion is unclear. To visualize the dynamics of ICAM-1 movement during adhesion, we have engineered stable Chinese hamster ovary cell lines expressing ICAM-1 fused to a green fluorescent protein (IC1_GFP/CHO) and examined them under the fluorescence microscopy. The transfection of IC1_GFP alone in these cells was sufficient to support the adhesion of K562 cells that express $\alpha$L$\beta$2 (LFA-1) integrin stimulated by CBR LFA-1/2 mAb. This phenomenon was mediated by ICAM-1-LFA-1 interactions, as an mAb that specifically inhibits ICAM-1-LFA-1 interaction (RRl/l) completely abolished the adhesion of LFA-1＊ cells to IC1_ GFP/CHO cells. We found that the characteristic of adhesion was followed almost immediately (~10 min) by the rapid accumulation of ICAM-1 on CHO cells at a tight interface between the two cells. Interestingly, ICI_GFP/CHO cells projected plasma membrane and encircled approximately half surface of LFA-1+ cells, as defined by confocal microscopy. This unusual phenomenon was also confirmed on HUVEC after adhesion of LFA-1＊ cells. Neither cytochalasin D nor 2,3-butanedione 2-monoxime an inhibitor of myosin light chain kinase blocked LFA-1-mediated ICAM-1 clustering, indicating that actin cytoskeleton and myosin-dependent contractility are not necessary for ICAM-1 clustering. Taken together, we suggest that leukocyte adhesion to endothelial cells induces specialized form of ICAM-1 clustering that is distinct from immunological synapse mediated by T cell interaction with antigen presenting cells.

• Journal of Ginseng Research
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• v.45 no.4
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• pp.490-497
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• 2021

Background and objective: Synthetic ginsenoside compounds G-Rp (1,3, and 4) and natural ginsenosides in Panax ginseng 20(S)-Rg3, Rg6, F4 and Ro have inhibitory actions on human platelets. However, the inhibitory mechanism of ginsenoside Rk1 (G-Rk1) is still unclear thus, we initiated investigation of the anti-platelet mechanism by G-Rk1 from Panax ginseng. Methodology: Our study focused to investigate the action of G-Rk1 on agonist-stimulated human platelet aggregation, inhibition of platelet signaling molecules such as fibrinogen binding with integrin α_IIbβ₃ using flow cytometry, intracellular calcium mobilization, fibronectin adhesion, dense granule secretion, and thromboxane B2 secretion. Thrombin-induced clot retraction was also observed in human platelets. Key Results: Collagen, thrombin, and U46619-stimulated human platelet aggregation were dose-dependently inhibited by G-Rk1, while it demonstrated a more effective suppression on collagen-stimulated platelet aggregation using human platelets. Moreover, G-Rk1 suppressed collagen-induced elevation of Ca²⁺ release from endoplasmic reticulum, granule release, and α_IIbβ₃ activity without any cytotoxicity. Conclusions and implications: These results indicate that G-Rk1 possess strong anti-platelet effect, proposing a new drug candidate for treatment and prevention of platelet-mediated thrombosis in cardiovascular disease.

• Journal of Applied Biological Chemistry
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• v.43 no.3
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• pp.131-135
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• 2000

Human polymorphonuclear neutrophils undergo diaphoresis after a selectin-mediated rolling on the endothelial cells of the blood vessel wall. Extravasation is believed to be an integrin-mediated process. Galectin-1 is a small dimeric beta-galactoside-binding protein synthesized by the endotherial cells and present in the perivascular connective tissue. In this study we suggest the possible role of galectin-1 in extravasation of the activated neutrophils. MAL lectin binding study showed, that f-MetLeuPhe-activated neutrophils decrease surface sialylation and increase galectin-1 binding via exposure of new galectin-1 binding sites. Desialylated HL-60 cells also show the same decrease in MAL binding and increase in galectin-1 binding, an increase which was not observed in the presence of lactose. Galectin-1 blotting analysis detected two possible major ligands (approximately 120 and 160 kDa) of galectin-1 from the desialylated HL-60 cell lysates.

• Journal of Acupuncture Research
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• v.20 no.5
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• pp.50-62
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• 2003

Objective: Bee Venom(BV) has been used for various kinds of inflammatory or painful conditions in Oriental Medicine clinics, and there publishes reports on its therapeutic effects and the probable mechanism of those therapeutic effects, where CDs and cytokines plays important role. This study investigated the influences of bee venom on the expressions of CDs and cytokines of HMC cell line Methods: In this study we analysed the expression profile of HMC cell line treated with BV of 10-2ug/ml in relation to that of HMC cell line treated with vehicle by way of CD/cytokine microarray hybridization with 342 genes on it. Results: There were no upregulated genes by more than 3 fold, while there showed some downregulated genes by less than 1/3 fold as follows: colony stimulating factor 2, CD122, IL-7, CD112, TNF-alpha, CD138, CD166, TGFbetaR2, CD42b, CD62L, CD111, interleukin 10 receptor alpha, colony stimulating factor 1(macrophage), CD38 antigen(p45), CD121a, CD33 antigen(gp67), colony stimulating factor 1 receptor, B cell linker protein (SLP65) mRNA, CD94, alanyl(membrane) aminopeptidase, immunoglobulin(CD79A) binding protein 1, CD205, CD241, CD207, CDw121b, integrin alpha L(CD11a), integrin beta 1(CD29), CD91, CD42b. Conclusions: Bee venom treatment induced downregulation of some CDs or cytokines including $TNF-{\alpha}$. IL-1R with its possible implication in an antiinflammatory action of BV. Further research on expression profile changes induced by BV treatment is expected.

• Journal of Radiopharmaceuticals and Molecular Probes
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• v.1 no.1
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• pp.46-52
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• 2015

Herein we present the synthesis of $^{68}Ga$-labeled gold nanoparticles for in vivo PET imaging. A novel chelator DTPA-Cys was easily prepared from diethylenetriaminepentaacetic dianhydride in high yield. The ${\alpha}_v{\beta}_3$ integrin receptor targeted gold nanoparticle probe was synthesized by using DTPA-Cys, polyethylene glycol and cRGD peptide. $^{68}Ga$ labeling of cRGD conjugated gold nanoparticle was carried out at $40^{\circ}C$ for 30 min. Observed radiochemical yield was more than 75% as determined by radio-TLC and the probe was purified by centrifugation. In vitro stability test showed that 90% of $^{68}Ga$-labeled gold nanoparticle probe was stable in FBS for 1 h. Those results demonstrated that $^{68}Ga$-labeled gold nanoparticle could be used as a potentially useful probe for specific tumor imaging.