• 한국수정란이식학회지
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• 제24권4호
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• pp.259-263
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• 2009

Salivary lipocalin (SAL1) is a member of the lipocalin protein family that has a property to associate with many lipophilic molecules and was identified as pheromone-binding protein in pigs. Our previous study has shown that SAL1 is expressed in the uterine endometrium in a cell type- and implantation stage-specific manner and secreted into the uterine lumen in pigs. However, function of SAL1 in the uterus during pregnancy in pigs is still not known. To understand physiological function of SAL1 in the uterine endometrium during pregnancy in pigs, it needs to elucidate the ligand(s) for SAL1. Thus, to identify the ligand for SAL1 in the porcine uterus, we collected uterine luminal fluid from pigs on day 12 of pregnancy by flushing with PBS. Proteins from the uterine luminal fluid were separated by ion exchange chromatography and gel filtration. Fractions containing SAL1 protein were pooled and concentrated. Immunoblot analysis confirmed successful purification of SAL1. Then, we extracted lipids from the purified SAL1 protein and analyzed the lipids by liquid chromatography-mass spectrometry, and predicted to be steroid hormones and prostaglandins as SAL1 ligands. Results in this study showed that SAL1 protein in the uterine secretions has a small lipophilic molecule as a natural ligand. Further characterization of ligand extracted from purified SAL1 will be useful for understanding physiological function of SAL1 during pregnancy and its application to increase the pregnancy rate in pigs.

• 한국미생물·생명공학회지
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• 제49권2호
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• pp.148-156
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• 2021

Single-chain antibodies against epidermal growth factor receptor variant III (EGFRvIII) are potentially promising agents for developing antibody-based cancer treatment strategies. We described in our previous study the successful expression of an anti-EGFRvIII scFv antibody in Escherichia coli. However, we could also observe the formation of insoluble aggregates in the periplasmic space, limiting the production yield of the active product. In the present study, we investigated the mechanisms by which growth conditions could affect the expression of the soluble anti-EGFRvIII scFv antibody in small-scale E. coli NiCo21(DE3) cultures, attempting to maximize production. The secreted scFv molecules were purified using Ni-NTA magnetic beads and protein characterization was performed using SDS-PAGE and western blot analyses. We used the ImageJ software for protein quantification and determined the antigen-binding activity of the scFv antibody against the EGFRvIII protein. Our results showed that the highest percentage of soluble scFv expression could be achieved under culture conditions that combined low IPTG concentration (0.1 mM), low growth temperature (18℃), and large culture dish surface area. We found moderate-yield soluble scFv production in the culture medium after lactose-mediated induction, which was also beneficial for downstream protein processing. These findings were confirmed by conducting western blot analysis, indicating that the soluble, approximately 30-kDa scFv molecule was localized in the periplasm and the extracellular space. Moreover, the antigen-binding assay confirmed the scFv affinity against the EGFRvIII antigen. In conclusion, our study reveals that low-speed protein expression is preferable to obtain more soluble anti-EGFRvIII scFv protein in an E. coli expression system.

• Biomolecules & Therapeutics
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• 제27권2호
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• pp.216-221
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• 2019

The c-Met protein is a receptor tyrosine kinase involved in cell growth, proliferation, survival, and angiogenesis of several human tumors. Overexpression of c-Met has been found in gastric cancers and correlated with a poor prognosis. Indirubin is the active component of Danggui Longhui Wan, which is a traditional Chinese antileukemic recipe. In the present study, we tested the anti-cancer effects of an indirubin derivative, LDD-1937, on human gastric cancer cells SNU-638. When we performed the in vitro kinase assay against the c-Met activity, LDD-1937 inhibited the activity of c-Met. This result was confirmed by immunoblot and immunofluorescence of phosphorylated c-Met. Immunoblot analysis showed that LDD-1937 decreased the expression of the Erk1/2, STAT3, STAT5, and Akt, downstream proteins of c-Met. In addition, LDD-1937 reduced the cell viability and suppressed colony formation and migration of SNU-638 cells. Furthermore, LDD-1937 induced $G_2/M$ phase arrest in the SNU-638 cells by decreasing the expression levels of cyclin B1 and CDC2. Cleaved-PARP, an apoptosis-related protein, was up-regulated in cells treated with LDD-1937. Overall, this study suggests that LDD-1937 may be a novel small-molecule with therapeutic potential for selectively inhibiting c-Met and c-Met downstream pathways in human gastric cancers overexpressing c-Met.

• 대한환경공학회지
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• 제31권9호
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• pp.735-742
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• 2009

척추동물의 뼈는 hydroxylapatite [$Ca_{10}(PO_4)_6(OH)_2$, HAp]로 이루어져 있어서 용액중에 존재하는 중금속이온들의 훌륭한 이온교환제로서 잘 알려져 있다. 본 연구에서는 하소시켜 얻은 소뼈, 돼지뼈, 생선뼈를 이용한 회분식 반응조에서의 구리이온의 제거성능을 조사하였으며 이러한 결과로부터 중금속이온을 제거하기 위한 흡착제로서의 재이용성을 평가하였다. 온도를 달리하여 하소시켜 얻은 세 종류의 뼈 시료들의 표면특성은 SEM, XRD 및 FT-IR 분석을 통하여 측정하였다. SEM 촬영결과 하소온도가 높아질수록 표면이 불규칙해졌으며 작은 기공이 발달하였다. XRD 분석에서는 하소 온도가 높아질수록 피크가 발달되는 경향을 보였는데 이와 같은 현상은 HAp가 열처리에 의해 무정형 형태에서 결정형으로 바뀌었음에 기인한 것으로 여겨진다. FT-IR 분석결과 하소온도가 높아질수록 흡착된 물과 골격 내 유기물의 흡수밴드의 강도는 점차 감소되는 것을 알 수 있었다. 구리이온 제거 결과 뼈를 넣지 않은 바탕실험에서는 15% 이하의 제거율을 보였는데 하소된 뼈를 사용한 경우에는 제거율이 부가적으로 40% 이상 상승하였다. 세 종류의 동물뼈 모두에서 하소온도를 높일수록 구리에 대한 제거율은 감소하였다. 용액의 pH가 상승할수록 양이온 교환 및 침전반응을 형성할 수 있는 유리한 조건에 의해 구리이온의 제거율이 상승하였다.

• Investigative Magnetic Resonance Imaging
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• 제2권1호
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• pp.73-82
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• 1998

목적 : 간특정 MR 조영제를 이요하여 간세포의 세포막을 통한 물분자의 교환 및 세포막 투과율을 정확히 측정 할 수 있는 MR 기법을 개발하고자 하였다. 대상 및 방법 : 쥐의 간세포를 분리하여 낸 후 NMR 측정을 시도하엿다. 모든 실험은 0.02MHz부터 60 MHz까지 양성자의 Larmor 주파수를 변화시킬 수 있는 IBM형 field cycling relaxometer를 사용하여 시행하였으며 spin-echo 펄스열을 사용하여 T1 자기이완시간을 측정하였다. 전오도의 간특정 조영제인 Gd-EOB-DTPA를 함유하고 있는 간세포 샘플로부터 획득한 T1 데이터를 연속분포 분석법을 사용하여 분석하였으며 이때 이론적 모델로는 Two compartmental exchange 모델을 이용하였다. 결과 : 간세포내의 물분자의 평균 거주시간은 약 250 msec이며 간세포막의 투과율에 대한 최저치는 $(1.3{\pm}0.1){\;}{\times}{\;}10^{-3}cm/sec$ 이었다. 자기이완시간의 연속적인 분포도를 구할 수 있는 CONTIN 분석기법을 적용한 결과 확산적 물분자 교환이 일어남을 밝혔고 이러한 확산적 교환의 정도가 간세포의 경우 세포내 공간에서는 작지 않다는 사실을 규명 할 수 있었다. 결론 : 연속분포 분석기법을 적용하는 경우 Gd-EOB-DTPA는 간세포에서의 물분자의 교환정도 및 세포막의 물분자에 대한 투과율을 측정하는데 매우 유용한 방법임을 확인하였고 간세포에서의 물분자의 교환속도는 적혈구에서의 물분자 교환 속도에 비해 매우 느리다는 사실을 확인하였다. 따라서 조직 특정 조영제는 해당 조직 혹은 세포의 세포막 투과율과 같은 생리학적 정보를 알아낼 수 있는 기능적 조영제로서의 유용성을 입증할 수 있었다.

• Journal of Chest Surgery
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• 제36권1호
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• pp.7-14
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• 2003

Cyclin E는 Gl-S기로의 이행을 조절하는데 매우 중요한 역할을 하는데, 세포주기 중 $G_{0}$ / $G_{1}$기로부터 S기로의 이행의 조절이상은 종양형성에 중요한 요소로 알려져 있으므로, 여러 종류의 악성종양에서 cyclin E의 발현은 종양의 생물학적 양상과 관련되어 보고되고 있다. 또 cdk2-cyclin E 복합체의 활성도는 cdk 억제인자인 p27$^{kip1}$의 분해에 의하여 증가한다. 그러나 비소세포폐암에서 cyclin E와 p27의 발현에 관한 연구는 매우 드물다. 대상 및 방법: 81례의 절제된 제1병기 비소세포폐암 조직을 이용하여 cyclin E와 p27의 발현율을 조사하고, 조직학적 유형, 분화도, 종양의 크기, 늑막의 침범 여부, 생존율과의 상호관계를 비교 분석하였다. Cyclin E와 p27의 발현은 특이한 단클론 항체를 이용하여 ABC법으로 면역조직화학염색을 시행하였고, 생존율은 Kaplan-Meier법을 이용하였다. 결과: 제1병기 비소세포폐암에서 cyclin E와 p27의 발현율은 각각 29.6%,28.4%였다. Cyclin E는 늑막 침범례에서, p27은 종양의 직경이 3cm 이하인 경우에 각각 높은 발현율을 보였다(p=0.04, p=0.015). Cyclin E의 발현군의 5년 생존율은 44.4%로, 미 발현군 68.2%에 비해 낮았으며(p=0.015), p27의 발현군의 5년 생존율은 72.2%로, 미 발현군 56.2%에 비해 높은 경향을 보였다(p=0.09). Cyclin E가 발현되지 않고 p27이 발현되는 군의 5년 생존율은 73.5%로, cyclin E가 발현되고 p27이 발현되지 않는 군 36.3%에 비해 높게 나타났다(p=0.0029). 다변량 분석상 cyclin E은 불량한 예후를(RR=3.578, p=0.006), p27은 양호한 예후를 시사하는 독립적인 인자였다(RR=0.183, p=0.009). 결론: 제1병기 비소세포폐암에서 cyclin E의 발현은 불량한 예후를, p27은 양호한 예후를 나타내는 독립적인 예후인자가 될 수 있음을 확인하였다.

• 대한후두음성언어의학회지
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• 제12권1호
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• pp.46-54
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• 2001

Background and Objectives : Nitric oxide(NO) is a short-lived molecule with messenger and cytotoxic functions in nervous, cardiovascular, and immune systems. Among the three distinct NOS isoforms, the neuronal isoform is expressed in small, discrete neuronal populations of CNS and PNS. Axonal injury in adult animals results in a dramatic NOS up-regulation in many types of central and peripheral neurons which normally lack the enzyme or express it only at very low levels. In previous study, we confirmed the efficacy of PEMS on the early functional recovery in rats with surgically transected and reanastomosed recurrent laryngeal nerve. Therefore, after we obtained functionally recovered rats using PEMS in this study, we studied to evaluate the expression of nNOS through the analysis of the difference between functional recovery group and non-recovery group in the recurrent laryngeal nerve. Materials and Method : Using 84 healthy male Sprague-Dawley rats, transections and primary anastomosis were performed on their left recurrent laryngeal nerves. Rats were then randomly assigned to 2 groups. The rats in group A(n=42) received PEMS by placing them in custom cages equipped with Helm-holz coils(3 hr/day, 5 days/wk, for 12 wk). The rats in group B(n=42) were handled the same way as the group A, except that they did not receive PEMS. Laryngovideoendoscopy was performed before and after surgery and followed up weekly. Laryngeal EMG was obtained in both PCA and TA muscles. Immunohistochemisty staining using monoclonal anti-neuronal nitric oxide synthase(nNOS) antibody was performed to detect nNOS in recurrent laryngeal nerve and nodose ganglion. Results : 20 rats(63%) in group A and 5 rats(17%) in the group B showed recovery of vocal fo1d motion. The number of NOS-positive cells was increased in functionally-recovered rats. NOS-staining intensity was reduced 12 weeks after nerve injury. The difference between PEMS group and non-stimulated group was not found. Conclusion : This study shows that nNOS may exert a beneficial effect on nerve regeneration and functional repair.

• Korean Chemical Engineering Research
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• 제49권5호
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• pp.669-675
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• 2011

다공성 구조로 되어있는 차세대 저유전 박막(k<2.0)의 나노 기공의 초기 형성 과정을 이해하기 위하여 실세스퀴옥산(silsesquioxane; SSQ) 매트릭스에서 분산된 4-tert-butyl calix[4]arene-O,O',O",O"'-tetraacetic acid tetraethyl ester(CA[4]) 포로젠이 열분해에 의해서 나노 기공으로 전환되는 과정을 Fourier Transform Infrared Spectroscopy(FT-IR)와 in-situ Position Annihilation Lifetime Spectroscopy(PALS) 연구를 통해 분석하였다. SSQ/CA[4] 하이브리드 시스템은 열 경화에 따라 효과적인 기공 구조의 균일한 박막을 제공하였다. SSQ/CA[4] 10, 20% 두 종류의 하이브리드 박막을 in-situ PALS 분석을 시행한 결과, CA[4] 포로젠의 분해 거동이 달랐다. SSQ/CA[4] 10% 하이브리드 박막은 $300^{\circ}C$ 이상부터 단분자 포로젠으로부터 기인한 메조포어(~1.5 nm)가 생성되기 시작하였으나, SSQ/CA[4] 20% 하이브리드 박막은 상대적으로 낮은 온도인 $250^{\circ}C$부터 상태로 CA[4] 분자들이 자가 조립된 마이셀로부터 기인한 메조포어(2.5~3.0 nm)가 생성되었다. 이는 SSQ/CA[4] 20% 하이브리드 박막에서 생성된 기공의 구조가 매우 연결된 상태이기 때문에 초기에 포로젠이 분해되었을 때, 분해된 분자조각들이 쉽게 박막 외부로 빠져나올 수 있기 때문이라고 생각된다.

• Journal of Periodontal and Implant Science
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• 제29권3호
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• pp.507-519
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• 1999

The ultimate objective of periodontal treatment is to stop disease progression and to regenerate destroyed periodontal tissues and thereby regain normal function. Growth factors are naturally found polypetides which stimulate many cellular activities pertaining to wound healing by acting as signal molecule in controlling cell movement, proliferation, and matrix production. Platelet derived growth factor (PDGF) is 28,000-35,000 Da molecular weight dimeric protein with 2 long positively charged polypeptide chains connected by sulfide bonds. The purpose of this study is to evaluate histologically the initial guided tissue regeneration in a periodontal defect f a beagle dog treated with a biodegradable membrane formed with polylactic acid (poly-L-lactic acid) and polyglycolic acid loaded with 200ng/$cm^2$ platelet derived growth factor. 2 beagle dogs were used in he experiment. $5mm{\times}6mm$ alveolar bone defect was formed in upper and lower canines and third premolars and a reference notch was placed. PDGF-BB non-containing membrane was used as control. Each defect was randomly assigned to the test roup or the control group. The dogs were sacrificed 3 weeks after membrane placement. Toluidine blue and multiple staining was done for histological analysis. In the 3 week specimen in the control group, no new one formation could be seen. Small amount f bone resorption below the notch could be seen. In the notch, loose connective tissue with infiltration of inflammatory cells could be seen. Also thin discontinuous new cementum could be seen and the membrane still retained its structure. Where PDGF-BB containing membrane was used, new bone formation could be seen in the notch at weeks and also continuous thin cementum could be seen. PDL cells were observed between new bone and new cementum and some were attached to bone and cementum. These results suggest that new bone and cementum formation seen when PDGF-BB loaded membrane was used was due to inhibition of downgrowth of epithelial cells and also due to continuous release of the growth factor. Further study on the resorption characteristics of the membrane nd the release characteristics of the PDGF-BB is necessary. Also, development of a membrane easier to use clinically is necessary.

• The Korean Journal of Physiology and Pharmacology
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• 제21권3호
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• pp.309-316
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• 2017

Transient receptor potential vanilloid 3 (TRPV3) is a non-selective cation channel with modest permeability to calcium ions. It is involved in intracellular calcium signaling and is therefore important in processes such as thermal sensation, skin barrier formation, and wound healing. TRPV3 was initially proposed as a warm temperature sensor. It is activated by synthetic small-molecule chemicals and plant-derived natural compounds such as camphor and eugenol. Schisandra chinensis (Turcz.) Baill (SC) has diverse pharmacological properties including antiallergic, anti-inflammatory, and wound healing activities. It is extensively used as an oriental herbal medicine for the treatment of various diseases. In this study, we investigated whether SC fruit extracts and seed oil, as well as four compounds isolated from the fruit can activate the TRPV3 channel. By performing whole-cell patch clamp recording in HEK293T cells overexpressing TRPV3, we found that the methanolic extract of SC fruit has an agonistic effect on the TRPV3 channel. Furthermore, electrophysiological analysis revealed that ${\gamma}$-schisandrin, one of the isolated compounds, activated TRPV3 at a concentration of $30{\mu}M$. In addition, ${\gamma}$-schisandrin (${\sim}100{\mu}M$) increased cytoplasmic $Ca^{2+}$ concentrations by approximately 20% in response to TRPV3 activation. This is the first report to indicate that SC extract and ${\gamma}$-schisandrin can modulate the TRPV3 channel. This report also suggests a mechanism by which ${\gamma}$-schisandrin acts as a therapeutic agent against TRPV3-related diseases.