• Microbiology and Biotechnology Letters
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• v.24 no.4
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• pp.472-477
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• 1996

Natural human epidermal growth factor (nhEGF) was purified from pregnant human urine by benzoic acid adsorption, DEAE-Sepharose ion exchange, and immunoaffinity chromatography. The purified nhEGF was further separated into four fractions using Bondapak C$_{18}$ HPLC system. Following characterization by Western blot analysis and double immu- nodiffusion, we found that each fraction corresponds to four derivatives of the nhEGF. For biological analysis of nhEGF, we optimized the labeling time and serum concentration for the incorporatioin of 5-bromo-2'-deoxy uridine (BrdU), a non-radioactive alternative for [$^{3}$H]-thymidine uptake, into NIH 3T3 cells. The DNA synthesis of NIH 3T3 cells was gradually increased at the nhEGF concentrations between 0.1 - 10 ng/ml in the Dulbecco's Modified Eagles Medium (DMEM) containing 0.2% Fetal calf serum (FCS). When we assayed the biological activity of four fractions, the activity of the second fraction was superior to that of the others. Based on the results from the HPLC analysis spiked with recombinant human epidermal growth factor (rhEGF) and amino acid sequencing, we concluded that the second fraction was nhEGF and the other three fractions were the derivatives of nhEGF. In addition, the proportion of nhEGF was approximately 46% is compared with that of the other three derivatives.

• YAKHAK HOEJI
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• v.40 no.6
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• pp.646-652
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• 1996

Epidermal Growth Factor (EGF), discovered by Stanley Cohen in 1960, has a potential healing effect for wounds and bums. Considering wound care, in order to avoid physical stress at the wound surface and efficiently apply EGF, the need for viscous spraying solutions was essential. Viscous spraying solutions containing EGF were prepared by utilizing viscosity-building polymer, poloxamer 407, and by introducing liposome systems. On the other hand, EGF is purified on reverse HPLC gradient program with the mobile solvent of acetonitrile. It is necessary to observe liposomal EGF changes as the acetonitrile contents varied in order to introduce liposome systems at the step of EGF solution (at the time of EGF purifying). By evaluating the size distribution and entrapment efficiency of EGF liposome, it was possible to detemine the limit contents of acetonitrile and establish the optimal conditions for solution formulations. It has been revealed that, as the acetonitrile content increases, mean diameter of EGF liposomes increased and the width of size distribution tends to decrease. The limit contents of acetonitrile were 10%, since there was little difference to the acetonitrile free liposomes.

• Microbiology and Biotechnology Letters
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• v.24 no.4
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• pp.478-484
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• 1996

Recombinant human epidermal growth factor (rhEGF) was produced by E. coli BL21 harboring a plasmid pYHB101. The maximum production was 68.7 mg/l when the E. coli strain was cultured at 25$\circ$C for 48 hours in the modified MBL medium containing 10 g/l glucose with 1 mM IPTG induction at 2 hours after inoculation. The rhEGF was purified upto 267 folds by Amberlite XAD- 7 chromatography, ultrafiltration, and DEAE Sepharose fast flow ion exchange chromatography with an overall yield of 66.6%. The purified rhEGF was further separated into two fractions by HPLC. The N-terminal amino acid sequence of the second fraction was Asn-Ser-Asp-Ser-Glu-Cys-Pro-Leu-Ser-His. The effect of rhEGF on the DNA synthesis was examined using in vitro biological assay based on the incorporation of 5'-bromo-2'- deoxy-uridine (BrdU). The purified rhEGF shows no difference with natural human epidermal growth factor (nhEGF) in N-terminal amino acids residues and biological activity. From the results, we concluded that rhEGF produced from E. coli harboring the plasmid pYHB101 was apparently the same as nhEGF.

• BMB Reports
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• v.32 no.1
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• pp.28-32
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• 1999

Annexin I (also called lipocortin 1), a 37-kDa member of the annexin family of proteins, has been implicated in the mitogenic signal transduction by epidermal growth factor (EGF). Annexin I is phosphorylated by the EGF signal, however, the role of annexin I in the EGF signal transduction is still unknown. To transduce extracellular signals into the intracellular targets, selective translocation of the signaling molecules to their targets would be necessary. In this study, we examined the subcellular locations of annexin I during EGF signal transduction. Treatment of A549 cells with EGF resulted in the translocation of cytoplasmic annexin I to the nucleus and perinuclear region as determined by Western blot and immunofluorescent staining. The nuclear translocation of annexin I was inhibited by tyrphostin AG 1478 and genistein, the inhibitors of EGF receptor kinase and downstream tyrosine kineses, respectively. Pretreatment of cells with cyclohexamide did not inhibit the nuclear translocation. The results suggest that nuclear translocation of annexin I is controlled by a series of kinase dependent events in the EGF receptor signaling pathway and may be important in tranducing the signals by EGF.

• Journal of Periodontal and Implant Science
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• v.26 no.4
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• pp.841-858
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• 1996

Epidermal growth factor(EGF) is one of polypeptide growth factors. EGF has been reported as a biological mediator which regulates activities of wound healing process including the cell proliferation, migration and metabolism. The purposes of this study is to evaluate the effects of EGF on the human periodontal ligament cells and human gingival fibroblast cells that promote regeneration of periodntal tissue. The mitogenic effects of epidermal growth factor on human periodontal ligament cells and human gingival fibroblasts were evaluated by determining the incorporation of 5-Bromo-2'-deoxy-uridine into DNA of the cells in a dose dependent manner. The prepared cells were the primary cultured gingival fibroblast and periodontal ligament cells from humans, the fourth or sixth subpassages were used in the experiments. Cells were seeded in DMEM containing 10% FBS. 1, 10, 50, 100, $200{\eta}g/ml$ and epidermal growth factor were added to the quiescent cells for 24 hours, 48 hours and 72 hours. They were labeled with $10\{mu}l/200{\mu}l$ 5-Bromo-2'-deoxy-uridine for the last 6 hours of each culture. The results of the five determinants were presented as mean and S.D.. The results were as follows : The DNA synthetic activity of human gingival fibroblasts were increased dose dependently by epidermal growth factor at 24 hours, 48 hours and 72 hours. The mitogenic effects were similar at the 24 and 48 hours of epidermal growth factor, but the DNA synthetic activity of human gingival fibroblasts generally decreased at 72 hours. The DNA synthetic activity of human periodontal ligament cells were increased dose dependently by epidermal growth factor at 24 hours but the DNA synthetic activity decreased at $200{\eta}g/ml$ of each hour. Generally the maximum mitogenic effects were observed at the 48 hours application of epidermal growth factor. The DNA synthetic activity of human periodontal ligament cells generally decreased lower at 24, 72 hours than at 48 hours the application of epidermal growth factor. In the comparison of DNA synthetic activity between human gingival fibroblasts and human periodontal ligament cells, human periodontal ligament cells had slightly higher proliferation activity than human gingival fibroblasts for a longer time at the high dosage of the epidermal growth factor. In conclusion, epidermal growth factor have important roles in the stimulation of DNA synthesis in human periodontal ligament cells and human gingival fibroblasts, and thus may be useful for clinical applications in periodontal regenerative procedures.

• Korean Journal of Animal Reproduction
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• v.20 no.2
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• pp.127-134
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• 1996

The objective of this experiment was to test the ability of the fertilization of EGF treated pig oocytes for in vitro maturation. The addition of EGF (10 ng/ml), FSH (10 ${\mu}\textrm{g}$/ml), or FBS (10%) on maturation medium of pig immature oocytes divided into four groups as follows; group 1: untreatment, group 2: EGF alone, group 3: combination of FSH and FBS, or group 4: combination of EGF, FSH, and FBS. The interactive effects of nuclear maturation rates (M II%) of EGF alone, FSH plus FBS, and EGF plus FSH added FBS treatments were significantly higher than those of non-treatments (P<0.001). The fertilization rate of EGF alone (group 2) was lower than that of 3, 4 groups, but was significantly higher than group 1 (p< 0.005). Furthermore, combination of EGF, FSH,and FBS (group 4) was higher than others (group 1. 2, 3) on male pronuclei formation as well as penetration of sperm (P<0.05). These results suggested that EGF alone decreased the ability of cytoplasmic maturation compared to nuclear maturation in pig oocytes, but a high level of cytoplasmic maturation of in vitro-matured pig oocytes can be achieved when supplemented with FSH and FBS.

• Korean Journal of Animal Reproduction
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• v.19 no.3
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• pp.217-226
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• 1995

The objective of this experiment was to test the effect of EG F on nuclear maturation of pig immature oocytes in vitro. Basic medium used TCM-199 supplemented with 0.2 mM pyruvate, 1 ${\mu}\textrm{g}$/ml estradiol-I7$\beta$ and 25 ${\mu}\textrm{g}$/ml gentamycin, this medium treated with EGF, FSH and FBS. Experiment 1 examined to the effect according to the addition of FSH or EGF (0, 1. 10 and 100 ng EGF/ml) in oocytes maturation. Nuclear maturation rates (M ll %) of 1, 10 and 100 ng EGF/ml (83.0. 8fi.7 and H7.5%) treatments were significantly higher than those of non- and FSH-treated groups (27.3 and 60.3%, p < 0. 001). Experiment 2 examined to the interactive effects of EGF. FSH or FBS during oocytes maturation. Nuclear maturation rates (M ll %) of EGF alone, EGF plus FSH, EGF plus FBS, FSH plus FBS, and EGF plus FSH added FBS treatments (86.7, 90.2, 87.1. 89.6% and 92.6%) were significantly higher than those of non, FSH, and FBS alone treatments (22.3, 52.2 and 42.3%, p < 0.001). Also, cumulus cells expansion of oocytes maturation was examined to total treatments. Normal cumulus cells expansion was shown by FSH plus FBS, EGF or EGF with FBS combination treatments, but cumulus cells of oocyte complexes were still clumped together in EGF-treated groups although they had separated from oocytes. However, EGF showed a positive on nuclear maturation. These results conclude that EGF alone can stimulate nuclear maturation in pig immature oocytes.

• Journal of Chest Surgery
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• v.34 no.2
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• pp.138-147
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• 2001

배경: 폐암발생에 EGF의 자가 분비는 암의 성장과정에 직, 간접적인 영향을 주고 있으며, TNF-$\alpha$는 면역 반응의 급성체로서 폐암의 발생을 억제하고 이미 발생한 폐암종의 치료에도 이용되고 있는 실정이다. 폐암 조직과 혈장에서 epidermal growth factor(EGF)와 tumor necrosis factor-$\alpha$(TNF-$\alpha$)를 면역 방사선 분석법을 이용하여 정량분석 하여 발현 정도를 분석해보고자 하였다. 대상 및 방법: 폐암환자 20례와 양성종양 및 육아종 환자 4례에 대해서 AJCCS에 의한 조직학적 분류와 TNM 분류에 따라 구분하여 절제수술을 받은 환자를 대상으로 수술전 혈액을 채취하고 수술직후 적출한 표본을 암이 없는 건강하다고 판단되는 대조조직과 폐암조직에서 일정량의 조직을 절취하여 액화질소 내에 실험시까지 급속 냉동보관 하였다. 수술후 혈액을 재 채취하여 혈장을 분리하여 냉동고에 검사시까지 보관하였다. EGF의 정량은 Human Epidermal Growth Factor kit(Amersham Phamacia Biotech, England)를 사용하였으며, TNF-$\alpha$ 정량은 TNF-$\alpha$ IRMA kit(Biosouce, Belgium)을 사용하여 IRMA 방법으로 각각 정량분석하여 표현유무를 연구한 결과 다음과 같은 결론을 얻었다. 결과: 1. 대조조직, 양성종양 및 육아종과 폐암 수술전후의 조직과 혈청 모두에서 EGF와 TNF-$\alpha$가 발현되었다. 2. EGF와 TNF-$\alpha$의 농도는 대조조직과 양성종양(0.11$\pm$0.06 ng/ml, 20,3$\pm$9.08 pg/ml)에 비하여 폐암조직(0.13$\pm$0.05 ng/ml, 34.34$\pm$47.74pg/ml)에서 유의하게 높은 농도가 발현되고 있었다. 3. 폐암중 선암조직에서 특히 TNF-$\alpha$(80.92$\pm$104.08 ng/ml)의 발현이 강하게 나타났다. 4. 혈청내의 EGF와 TNF-$\alpha$의 발현되는 양이 조직내의 양보다도 높았다. EGF는 5.7배정도 TNF는 1.3배정도 강하게 표현되었다. 5. 폐암의 조직학적 종류에 따라서 EGF는 거의 차이가 없었으나 TNF-$\alpha$ 정량치에는 차이가 있었다. 6. TNM stage가 진행함에 따라 EGF는 농도가 증가하였고 TNF-$\alpha$는 오히려 감소하는 반대되는 교차현상이 있었다. 7. 수술직후 EGF는 증가하였으나 TNF-$\alpha$는 오히려 감소하였다. 결론: 결론적으로 저자는 암조직과 대조조직간에 EGF와 TNF-$\alpha$의 표현량의 차이가 있음을 관찰하였으며 또한 조직과 혈청사이에도 표현량에 차이가 있으며 조직보다도 오히려 혈청내의 농도가 높다는 사실을 관찰하였다. EFG와 TNF-$\alpha$는 정상조직이나 양성조직과 폐암조직 모두에서 분비작용되는 cytokines으로 세포기능에 따라 다양하게 표현이 되며 계속적인 연구로서 밝혀야만 할 과제라고 판단된다.

• The Korean Journal of Zoology
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• v.39 no.2
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• pp.215-222
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• 1996

In the previous studies, we have demonstrated that prorenin converting enzyme (PRECE) was identical to the epidermal grouch factor-binding protein (EGF-BP) type B, which was a member of the mouse glandular kallikrein family, To examine whether or not EGF-BP type A and C are involved in the processing of prorenin, we have cloned the CDNAS of the EGF-BP type h and C from a library of male ICR mouse submandibular gland (SMGI. And then CHO cells were transfected with the EGF-BP expression plasmids. and stable cell lines expressing a high level of the EGF-BPS precursor were obtained. The conditioned medium was then treated with trypsin, which has been knotvn to effectively convert the EGF-BP type A and C precursor to the active forms. 수ubsequentlv, the prorenin converting activity of the trypsin-treated or untreated medium was examined. PRECE converted exactly prorenin to renin, but the prorenin converting activities of EGF-BP type A and C were not detected. From these results, it seems that only type B of these EGF-BPs is involved in processing Ren 2 prorenin in mouse SMG.

• Archives of Pharmacal Research
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• v.24 no.4
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• pp.355-359
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• 2001

A novel HPLC method with electrochemical detection has been developed for the determination of recombinant human epidermal growth factor (rhEGF) in pharmaceutical products. rhEGF was separated from other components in formulation on a reversed-phase C18 column with 24% acetonitrile in 0.1 M phosphate buffer (pH 4.75). The optimum electrochemical oxidation of EGF was obtained at 0.85 V vs. Ag/AgCl in a glassy carbon working electrode due to electroactive tyrosine, tryptophan, methionine, and arginine residues. The quantitation range was from 1.0 to 200 ng of rhEGF with the linear correlation coefficient greater than 0.999. The method was successfully applied for the quantitation of rhEGF in a pharmaceutical preparation.