Glucocorticoid inhibition of antigen - induced inositolphosphate formation by basophilic leukemia cells : possible involvement of phosphatases and other several proteins.

허억(Erk Her) 건국대학교 유전공학연구소 1993 제1회 건국대학교-KIST유전공학연구소 공동심포지움 Vol.- No.-

초내식성 스테인레스강의 금속이온용출특성 및 세포적합성

김철생,박진수,허억,강곤,Kim, Cheol-Sang,Park, Jin-Soo,Her, Erk,Khang, Gon 대한의용생체공학회 1996 의공학회지 Vol.17 No.1

The toxic metal ion release behaviour and the cytotoxicity of a super stainless steel (S.S.S, 22cr-20Ni -6Mo-0.25N) were investigated The measurement of the amount of static and wear- induced trace metal ion released from the steels was conducted in Hank's balanced salt solution using an electrothermal atomic absdrption spectrometry equiped with Uaphite furnace. And the in vitro cytotoxicity of the materials was assesed in cell culture. The static dissolution rates of Fe and Cr ions from the S.S.S were significantly lower than those of 316L SS. However, the Ni ion release from the S.S.S during the first 4 weeks was yester than that from 316L 55 by 15-45%. Also, the wear-in- duces dissolution rates from the steels were not correlated either with their elemental composition rates or with the static metal ion release rates. The S.S.S did not deteriorate the osteoblasts viability. And no toxic response was observed from the macrophages cultured for 7 days in RFMI 1640 medium immersed with the S.S.S specimens.

홍삼(紅蔘), 황기, 감국(甘菊) 혼합물(混合物) 제재 효과에 관한 연구

성현제,정종운,류충열,허억,Sung Hyun-Jea,Chung Chong-Un,Ryu Choong-Yul,Her Erk 대한한의학방제학회 2004 大韓韓醫學方劑學會誌 Vol.12 No.1

1. All the male students and female students who had taken these herb medicines showed a strong increase in immunity (P<0.01) and they were more effective to the male students (P<0.01) rather than the female students (P<0.05) (figure 1). Generally, immunity may be reduced at the time of fatigue and overwork but as these herb medicines have the effect of increasing immunity. Therefore, it is considered that they not only reduce the fatigue of human body but also heighten immunity against disease. 2. The students who had taken these herb medicines showed some reduction in Cortisol concentration in blood, which didn't show the difference of concentration up to statistical meaning compared to before taking the medicines (figure 2). Accordingly, it is regarded that these herb medicine drink restrained stress slightly. 3. The students who had taken these herb medicines showed some reduction in BUN concentration in blood, which didn't show the difference of concentration up to statistical meaning compared to before taking the medicines (figure 3). For the women, they restrained the toxicity of the kidneys just a little. Accordingly, it shows that these medicines are safe herb medicines without the toxicity of the kidneys. 4. The students who had taken these herb medicines didn't show the difference of concentration up to statistical meaning in sFOT and sGPT concentration in blood compared to taking them (figure $4{\sim}5$). Accordingly, it is suggested that these medicines are safe stuffs without the toxity of the liver. 5. Seven members of the university students answered to the questionaire that they were pleasant while taking the medicines and didn't feel fatigue. They answered very positively that they had better effects than before taking the medicines in many things (Refer to the Questionaire). However, one male student and two female students of them complained of temporary headache and indigestion for 2 days after taking these herb medicines but they said they had been taking cold medicines due to cold symptom before taking the medicines. Accordingly, it turns out that such temporary symptom is not irrelevant to these medicines. In conclusion, it is considered that these herb medicines have the effects of increasing immunity, restraining fatigue and stress through this clinical study carried out in simple way and that they are valuable as herb medicines without the toxity of the liver and kidneys.

Roles of Src-family kinase isoforms, Lyn, Fyn, Fgr, and c-Src on degranulation in RBL-2H3 mast cells

이준호,문세환,고나영,김지완,김도균,김주동,허억,최완수,Lee, Jun-Ho,Mun, Se-Hwan,Ko, Na-Young,Kim, Jie-Wan,Kim, Do-Kyun,Kim, Joo-Dong,Her, Erk,Choi, Wahn-Soo Korean Society of Life Science 2007 생명과학회지 Vol.17 No.3

흰쥐유래의 비만세포인 RBL-2H3 세포는 다양한 Src-family kinase를 발현한다. 현재까지의 연구결과에 의하면 비만세포의 초기 활성화에 Lyn kinase가 중요한 역할을 한다고 알려져 왔다. 그러나 그 세포에서 발현되는 다양한 다른 Src-family kinase의 역할은 불분명하다. 본 연구에서는 비만세포에서 다양한 Src-family kinase가 세포내 다른 곳에서 다양하게 발현되고 있다는 사실을 RT-PCR, immunoblotting 그리고 confocal microscopy 기법을 이용하여 증명하였다. 그 결과 Lyn 및 Fgr kinase는 세포막에 위치하고 c-Src 및 Yes kinase는 세포 내 과립에 존재하는 것을 알 수 있었다. 모든 Src-family kinase를 클로닝하고 과발현하여 탈과립 대한 영향을 평가하였다. 그 결과 fyn과 Fgr kinase는 비만세포에서 항원 유도의 탈과립을 증가시켰으며 반면 Lyn kinsae는 탈과립을 억제시키는 것을 확인할 수 있었다. 이러한 결과는 비만세포 초기 신호전달계에서 Fgr가 중요한 역할을 할 가능성을 제시한다. The rat RBL-2H3 mast cells contain various Src-family kinases. Previous reports with this cell line indicated that Lyn activation is an important initial signaling for the activation of the cells. However, the role and location of other Src-family kinase isoforms which are expressed in the cells are not clear. In this study, we now show that isoforms of Src-family kinases, Lyn, fyn, Fgr, c-Src, and Yes are differentially expressed and located differently in the cells as indicated by RT-PCR, immunoblotting analysis, and confocal microscopy. Lyn and Fgr were located on plasma membrane but on the other hand c-Src and Yes were located on intracellular organelle. All of Src-family kinases were cloned and overexpressed for investigating the roles of the isoforms. Overexpression of Fyn and Fgr, not Lyn and c-Src, stimulated Ag-induced degranulation in the cells. Our findings strongly suggest for the first time that each of Src-family kinase isoform can regulate differentially $Fc{\varepsilon}RI$-mediated signaling in RBL-2H3 mast cells.

Macrophages로부터 IL-1β 분비 및 전사에 있어서 한국산 겨우살이 추출물 M11C (non-lectin components)의 효과

장성호,전명하,강태봉,문세환,이준호,성낙술,이성태,김종배,허억,Chang, Sung Ho,Jun, Myung Ha,Kang, Tae Bong,Mun, Se Hwan,Lee, Jun Ho,Seong, Nak Sul,Lee, Sung Tae,Kim, Jong Bae,Her, Erk 대한면역학회 2001 Immune Network Vol.1 No.2

Background: Korean mistletoe (Viscum album) extract has been found to posses immunostimulatory activity. In this study, Korean mistletoe extract, M11C (non-lectin components), was used to know whether this extract might activate mouse peritoneal macrophages to produce interleukin $1{\beta}$ (IL-$1{\beta}$). Methods: Hemagglutination assay was carried out to examine whether M11C contained a lectin or not. To know the effect of M11C on the production of IL-$1{\beta}$, the macrophages were treated by the M11C, and then collected the supernatant (M11C stimulated macrophages-conditioned media; MMCM). MMCM was analyzed for the IL-$1{\beta}$ quantification and mRNA expression by means of ELISA and RT-PCR, respectively. Results: Maximum effective dose and time of M11C on IL-$1{\beta}$ production from macrophages were $20{\mu}g/m{\ell}$ and 8 hours, respectively. This ELISA data was reconfirmed by immunoblotting assay. indicating that M11C is a good candidate for an immunomodulator. The dose and time dependent effects of M11C on the expression of IL-$1{\beta}$ mRNA from macrophages was also shown in expression of mRNA detected by RT-PCR. Treatment dose and time for the maximum expression of IL-$1{\beta}$ mRNA were $20{\mu}g/m{\ell}$ and 4 hours, respectively. Maximum gene expression of IL-$1{\beta}$ was much earlier than maximum production of it. Conclusion: As results, Korean mistletoe extract, M11C, may be used for an immunomodulator. This will be able to make up for and solve the problems caused by existent immunoagent with many adverse effects through many other studies in future including one molecule extraction.

쥐의 비장세포로부터 IL-1β 분비에 있어서 한국산 겨우살이 추출물 M11C (비렉틴 구성물질)의 효과

강태봉(Tae Bong Kang),장성호(Sung Ho Chang),최완수(Wahn Soo Choi),성낙술(Nak Sul Seong),허억(Erk Her),전명하(Myung Ha Jun) 한국약용작물학회 2007 한국약용작물학회지 Vol.15 No.1

한국산 겨우살이 추출물 M11C (렉틴 구성물질)가 단구세포의 TNF-α 유전자 발현유도 및 분비에 미치는 효과

전명하(Myung Ha Jun),강태봉(Tae Bong Kang),장성호(Sung Ho Chang),최완수(Wahn Soo Choi),성낙술(Nak Sul Seong),허억(Erk Her) 한국약용작물학회 2007 한국약용작물학회지 Vol.15 No.1

PHA로 자극된 T cell에서 분비된 미지의 물질이 지니는 Human Neutrophil의 수명연장, Superoxide 및 Leukotriene C₄생산증가 작용효과

허억,양영목 大韓免疫學會 1993 大韓免疫學會誌 Vol.15 No.-

There are several direct and indirect ways in which T cells could enhance the anti-bacterial capabilities of neutrophils. However it is not yet clear out which molecule or cytokine produced by T cells is involved in the phagocytic action and viability sustaining activity of neutrophils. The aim of this study was about a factor, which produced by the phytohaemagglutinin(PHA)-stimulated T cells, may control those neutrophil actions. Human peripheral blood T cells and neutrophils were isolated by Ficoll-paque density sedimentation from heparinized blood of healthy adult donors. The purity of these cells were more than 90%. T cells were stimulated in various dose(0.1-10gg/ml) of PHA for various times of incubation(0-3 days), and then PHA-stimulated T cell conditioned medium was collected in order to find an optimal dose and incubation time for the neutrophil viability. It was found out that 1,ug/ml of PHA in 12 hours incubation was maximal effective condition for the neutrophil sustaining viability. The effects of PHA-stimulated T cell conditioned medium(TCM) on the neutrophils were used for the comparison with PHA-nonstimulated TCM or enriched medium alone. Neutrophil sustaining viability with PHA-stimulated TCM for 24 hours incubation was significantly higher than other groups (80 ± 10 vs 25 ±15 vs 13 ± 9; p <0.01). The superoxide prodution from neutrophils with PHA-stimulated TCM for 24 hours incubation were also significantly higher than other groups(25±4 vs 11±4 vs 7±5; p <0.01). In the leukotriene C4 (LTC4) release, neutrophils with PHA-stimulated TCM for 24 hours incubation were different from other group (105 ± 20 vs 65 ±1O vs 25 ± 32 ; p <0.01), and unlikely other parameters the cells with PHA-nonstimulated TCM was different from the cells with enriched medium alone(65 -1:10 vs 25-± 32; p<0.05). In two dimension electrophoresis it was shown that PHA-stimulated T cells enhanced three proteins(66kD, 60kD, 45kD) and diminished one(40kD) in the production and/or release of proteins in comparison with PHA-nonstimulated T cells. These data suggest that these proteins from the PHA-stimulated T cells might be involved in the phagocytic actions of neutrophils.

Human Neutrophil의 수명연장과 Superoxide 생산에 관여하는 미지의 Monocyte 생성물질

허억,배진우 大韓免疫學會 1993 大韓免疫學會誌 Vol.15 No.-

It has long been known that neutrophils are quickly infilterated and recruited to infected sites and then kill invaders by phagocytic action. Unfortunatly it is not yet revealed which molecule or cytokine is involved in the phagocytic action and viability sustaining activity of neutrophils. The aim of this study was whether lipopolysaccharide (LPS)-stimulated monocyte may control those neutrophil actions. Human peripheral blood monocytes and neutrophils were isolated by Ficollpaque density sedimentation from heparin anti-coagulated blood of healthy adult donors. After preparation of these cells, the purity of both was more than 90%. Monocytes stimulated in various dose(0.1-10pug/ml) of LPS for various times of incubation(0-3 days). and then LPS-stimulated monocyte conditioned medium was collected in order to find an optimal dose and incubation time for the neutrophil viability. It was found out that 3,ug/ml of LPS in 24 hours incubation was maximal effective condition for the activity of neutrophil sustaining viability. Monocyte conditioned medium (MCM) under this condition was used for the comparison with LPS-nonstimulated monocyte conditioned medium or enriched medium alone. When neutrophils were stimulated with each medium for 1-3 days, the activity of neutrophil sustaining viability with MCM was significantly higher than the activity with other medium (in 1 day of culture, 72-1:8 vs 4311:7 vs 17 ±10; p <O. 01). The superoxide production of neutrophil stimulated with MCM for 24 hours incubation was significantly higher than that with other medium under fMLP doses of 0.1-100,uM (p <0.01). Under fMLP l,uM, the superoxide production is predominantly different between them(23.8±2 vs 10.3±3 vs 7.8±1.6). The maximal effective dose of GM-CSF(granulocyte/macrophage colony stimulating factor ; 10pM) enhanced the neutrophil viability in 1 day of culture (50 ± 4%) . In the study to assess whether MCM contains GM-CSF, anti-GM-CSF antibody slightly blocked the MCM-dependent neutrophil viability(73±9 vs 50±12; p <0.07), indicating that MCM might not contain GM-CSF. These data indicate that LPS-stimulated monocyte surely product a factor for the neutrophil sustaining viability and the enhancement of superoxide production, suggesting that a factor is not GMCSF. If more than one factor were producted form LPS-stimulated monocyte, one minor factor might be GM-CSF.

A Dual Effect of Hydrocortisone on the Inhibition of intracellular Free Calcium Level Which are Highly Correlated with Leukotrien C₄ Production in Rat Basophilic Leukemia Cells : 세포내 칼슘량과 LEUKOTRIENE C₄ 생산은 不可無의 相關關係

Her, Erk 건국대학교 1992 學術誌 Vol.36 No.2

人造 부신피질 호르몬중의 하나인 HYDROCORTISONE(HC)은 가장 강한 抗 Allergy 抗염증약이다. 이 약은 Allergy와 염증을 일으키는 물질들의 생산과 세포의 분비를 억제하는 것으로 알려져 있다. Leukotriene(LTC4)는 이러한 Allergy와 염증을 일으키는 주요한 물질중의 하나이다. LTC4는 5-lipoxygenase(5-LOX)라는 효소에 의해 세포막 지질에서 생성된 Arachdonic acid라는 지방산으로부터 생산된다. 이 효소의 촉매작용은 칼슘의 농도에 크게 의존한다. 이러한 학문 바탕 위에서, 호중구 암세포(RBL-2H3)에 있어 항원에 의해 유기된 LTC4 생산을 HC가 어떠한 mechanism속에서 억제하는지 알고자 함이 실험연구의 목적이다. 기본실험에 있어, LTC4 생산을 의한 DNP10BSA 항체 IgE와 항원 DNP10BSA의 최고 효과적인 농도는 각각 2㎍/106 cells, 18.8ng/106 cells이었다. HC가 항원이 유기 생산한 LTC4량을 억제하는 것은 배양시간과 농도에 의존했었다. 최고 효과적인 항원 농도에 의해 유기 생산된 LTC4량을 50% 억제하는데 최고 효과적인 HC농도(1㎍/ml)로 2.5 시간의 사전 배양이 요구되었다. HC 사전 배양시간이 증가함에 따라 항원에 유기생산된 LTC4량이 감소하는데, 4시간 사전 배양은 control에 비해 75%을 감소시켰다(p < 0.005). 4시간 HC 사전 배양후, LTC4 생산을 억제하는데 HC의 IC50價는 0.3μM이였고, 최대 억제농도는 27μM였다. LTC4량을 측정하는데 Radioimmunoassay(RIA)법의 신뢰 여부를 알기 위해, 다른 방법중의 하나인 Thin layer chromatography(TLC)법을 이용하여 비교해 봤다. 비교 결과, RIA법의 신뢰도가 높았고, 上同 2개의 방법에 의한 값이 거의 相同했었으며, RBL-2H3 암세포는 LTC4를 거의 생산치 않고 주로 LTC4를 생산한다는 것이 발견되었다(LTC4 vs LTD4 p<0.005). 세포내 방사선 동위원소 칼슘(45Ca2+)의 침투량을 측정하는 실험에 있어서는 세포내의 칼슘의 이온화와 불이 온화를 구별할 수 없는 단점이 있어 이온화된 칼슘만을 측정할 수 있는 방법을 모색했었다. 세포내 이온화된 칼슘 측정이 중요한 것은 세포학에서 말하는 주요한 second messenger가 이온화된 칼슘이기 때문이다. 그래서 이온화된 칼슘의 양의 증감을 측정하기 위해 Quin2/AM법을 사용했다. 항 DNP10 BSA 항체 IgE가 감작된 RBL-2H3 암세포에 항원 DNP10BSA로 유기했을 때 세포내 이온화된 칼슘([Ca2+]i,)의 양이 비교구보다 3.5배나 증가했었다(p<0.01). 항원 DNP10 BSA로 RBL-2H3를 유기했을 때 [Ca2+]i량이 최고치에 도달하는 데 소요되는 시간은 1.5∼2분 정도였다. 그런데 HC가 사전 처리한 세포에서는 [Ca2+]i, 최고치가 현저히 억제되었다(p<0.01). 항원을 투입한 뒤 12분후, HC를 사전 처리하지 않는 세포에 있어서는 최고치량에서 7 ± 3% 정도 감소했었다. 이에 반해 HC를 사전 처리한 세포에서는 75 ± 7% 감소했었다(p<0.005). HC를 6시간 사전 처리한 세포들은 현저히 LTC4 생산뿐만 아니라 [Ca2+], 증가를 현저히 감소시켰다(p<0.05). 이러한 HC 효라는 세포내 칼슘 침투를 억제하는 藥 Nifedipine과 세포내 칼슘을 세포외로 pumping하는 藥 TPA의 효과와 흡사했다. 그래서 HC는 세포내 칼슘량을 억제하는 데 양면성이 보인다고 예측되어진다. 그리고 [Ca2+]i량의 증감과 LTC4 생산 증감과 밀접한 상관관계가 있다는 것이 통계적으로 입증되었다(r = 0.94, p<0.01). 결론적으로 호염기구에서 HC이 LTC4를 억제하는 mechanism은 일차적으로 미지의 단백질을 생산하고 이러한 단백질이 세포내 칼슘의 침투를 억제하거나 세포내 칼슘을 세포외로 pumping함으로써 [Ca2+]i량을 억제하는 것 같다. Hydrocortisone(HC) is extremely potent anti-allergic and antiinflammalory druge which suppress the biosynthesis and/or release of inflammatory mediators. Leukotriene C4 (LTC4) is one of the maim mediators, which is produced through the 5-lipoxygenase. This enzyme activity is dependent on high Ca2+ concentrations. The aim of this study was to insight the mechanism of HC actions on antigen-induced LTC4 formation in rat basophilic leukemia(RBL-2H3) cells. The maximal effective doses IgE and its antigen DNP10BSA for stimulation of the LTC4 formation were 2㎍.106 cells and 18.8ng/106 cells, respectively, The inhibitory effect of HC on the antigen-induced LTC4 formation was time-and dele-dependent. The half maximal effective time for the HC action was about 2.5 hr(hours) of preincubation. This time-dependent inhibition increased slowly to approximately 75% inhibition(p < 0.005) after 4 hr of pretreatment. Under 4 hr pretreatment with HC, IC50 values of HC on LTC4 production was 0.3μM, and maximal inhibitory dose was 2μM. In validation of the radiommunoassay(RIA) for the measurement of LTC4 production using a thin layer chromatography(TLC), I found that RBL-2H3 produced mainly LTC4, while only slightly LTD4 (LTC4 vs LTD4 p,0.005). The results obtained with TLC with regard to LTC4 formation were similar to that obtained by RIA. Since radioisotope labelled calcium(45Ca2+) studios have limited resolution to distinguish intracellular free calcium([Ca2+]i) from the whole cellular Ca2+, 1 measured [Ca2+]i, using a Quin2/AM probe. In the cells stimulated by maximal effective dose of antigen, HC markedly suppressed [Ca2+]i elevation (p 01). Half maximal time for elevation of [Ca2+]i induced by antigen was less than 1 min, and maximal elevation of [Ca2+]i (3 fold increase) was reached within 1.5∼2 minutes(min). This peak of [Ca2+]i level in the control group decreased gradually to 7 ± 3% during 12 min of incubation. Following pretreatment with HC, the antigen-stimulated increase of [Ca2+], was stunted by 41 ± 8%(p<0.01) after 2∼3 min and drastically reduced by 75 ± 7%(p<0.005) at 12 min. Preincubation for 6 hr of RBL-2H3 cells with HC(1㎍/ml) abolished the anti DNP10BSA antibody IgE and antigen DNP10BSA complex(Ag-Ab) induced [Ca2+]i increment and LTC4 production(p <0.05). This HC effect on two parameters mimicked the direct effects of Ca2+ efflux enhancer, TPA, and a Ca2+ channel blocks, nifedipine. Moreover, there was high correlation between [Ca2+]i elevation and LTC4 formation(4 = 0.94, p < 0.01), suggesting a causal relation between the two parameters. These data indicate that the inhibitory effect of HC on LTC4 formation requires a DNA-dependent protein synthesis and reduction of [Ca2+]i.