Mutation of individual protein C cleavage sites in Human coagulation factor Va

김성욱(Suhng Wook Kim),이승관(Suhng Wook Kim),이창규(Suhng Wook Kim) 고려대학교 보건과학연구소 2004 보건과학논집 Vol.30 No.1

  Resistance to activated protein C (APC) is the most common inherited risk factor for venous thrombosis. In the present study we have investigated the effect of mutations of the APC cleavage sites at Arg<SUB>306</SUB>, Arg<SUB>506</SUB> and Arg<SUB>679</SUB> in the factor V heavy chain on the inactivation of factor Va by APC. Mutants R306A, R506Q and R679A were expressed using B-domain deleted factor V constructs in COS-7 cells. The specific activity of the purified mutant proteins was identical to the wild-type protein (rHFVa) and APC cleavage at the mutated sites was blocked. Characterization of these mutants using a clinical assay for APC resistance demonstrated that R506Q was resistant to APC, but that R306A and R679A were sensitive. In clotting assays, R506Q showed delayed inactivation by APC, whereas R306A showed a rapid but incomplete loss of activity. APC inactivation of R679A was rapid and indistinguishable from rHFVa. These results indicate that mutation of individual APC cleavage sites in factor Va leads to variable degrees of APC. These findings have significant implications for the identification of patients with APC resistance and the understanding of the importance of individual APC cleavage sites in regulating the activity of the prothrombinase complex.

경쟁적 ELISA를 이용한 도파민과 세로토닌의 검출

남궁수민,최정수,박지향,양만길,이민우,김성욱,Namkung, Su Min,Choi, Jeong Su,Park, Ji Hyang,Yang, Man Gil,Lee, Min Woo,Kim, Suhng Wook 대한임상검사과학회 2017 대한임상검사과학회지(KJCLS) Vol.49 No.3

Dopamine (DA) and serotonin (5-Hydroxytryptamine, 5-HT) are neurotransmitters and hormones that exist in small amounts but have important role in the body. Serum and 24-hour urine are used as specimens, and are usually examined by HPLC-MS. In this study, we tried to detect DA and 5-HT by competitive ELISA using antigen-antibody (Ab) reaction. After immobilizing $5{\mu}g/mL$ BSA conjugate on a 96-well surface, hormone and primary Ab, which are respectively diluted to different concentrations, were treated. Then, HRP-conjugated secondary Ab and TMB were added to measure absorbance. The regression equation and $R^2$ value were calculated based on absorbance, and sensitivity of Ab to hormone as well as the correlation between hormone concentration and absorbance were determined. In DA ELISA, $R^2$, the correlation between the concentration of hormone and absorbance, was the highest by 0.91 when anti-dopamine Ab was diluted 6,000 times and 7,000 times. In 5-HT ELISA, $R^2$ was bigger than 0.90 in every concentration except 3,000 times and 6,000 times. Both DA and 5-HT were not effectively detected at low concentrations (less than $1.0{\times}10^{-7}M$); and because reference value of serum DA is lower than this, HPLC-MS was required to detect serum DA. However, competitive ELISA may be effective in detecting 24-hour urine DA, serum, and 24-hour 5-HT. Further studies are needed to detect hormones more accurately at lower concentrations.

연습용 10-20 Electrode System을 이용한 뇌파검사 실습의 효율성과 만족도 평가

이창희,김대진,최정수,이종우,이민우,조재욱,김성욱,Lee, Chang Hee,Kim, Dae Jin,Choi, Jeong Su,Lee, Jong-Woo,Lee, Min Woo,Cho, Jae Wook,Kim, Suhng Wook 대한임상검사과학회 2017 대한임상검사과학회지(KJCLS) Vol.49 No.3

Electroencephalography (EEG) is distinct from other medical imaging tests in that it is a functional test that helps to diagnosis disorders related to the brain, such as epilepsy. The most important abilities for a medical technologist when performing an EEG are knowing the exact location of the electrode and recording the EEG wave clearly, except for artifacts. Although theoretical education and practical training are both included in the curriculum for improving these abilities, sufficient practical training has been lacking due to problems like expensive equipment and insufficient practical training time. We try to solve these issues by manufacturing the training 10-20 electrode system and by estimating the efficiency and satisfaction of the training 10-20 electrode system through a questionnaire. The time required for practical training using this system was $43.58{\pm}9.647min$, which proved to be efficient. The satisfaction score of participants who experienced curriculum practical training was improved from $7.21{\pm}2.285$ to $9.46{\pm}1.166$. Based on these findings, it is considered that practical training via the use of the training 10-20 electrode system will solve the problems, such as lack of equipment and insufficient practical training time. Nonetheless, to further improve the training 10-20 electrode system, it must overcome the limitations of developing a device capable of checking the actual brain waves and validating the exact location of electrode attachment.

게르마늄 농도를 증가 시킨 동충하초 균사체 열수 추출액의 항암효과

최정수 ( Jeong Su Choi ),허지혜 ( Ji Hye Heo ),김대진 ( Dae Jin Kim ),남궁수민 ( Su Min Namkung ),이태복 ( Tae Bok Lee ),이민우 ( Min Woo Lee ),김성욱 ( Suhng Wook Kim ) 대한임상검사과학회 2017 대한임상검사과학회지(KJCLS) Vol.49 No.2

게르마늄(Ge)은 거의 대부분의 영양분, 동물, 식물에서 발견되는 미량원소로 많은 연구를 통해 항균성 항바이러스성 항종양성 항돌연변이성 및 면역조절 효과를 지닌 것으로 알려져 있다. 또한, 동충하초는 중국의 전통적인 약용 버섯으로 항종양성, 항산화성 및 면역조절 효과 등 다양한 생물학적 활성을 지니고 있으며 미량원소를 축적시키기 효과적인 것으로 알려져 있다. 이런 특성을 이용하여 본 연구는 게르마늄 농도를 증가시킨 동충하초(CMGe)의 열수 추출액을 얻어내고 그 생물학적 특성을 실험하였다. 네 가지 암세포주 Ramos, A549, HepG2, CEM와 MSC에 대한 CMGe의 세포독성을 MTT assay를 통해 확인한결과, CEM에서 항증식성이 가장 높고 MSC에 대한 세포독성은 낮은 것으로 확인되었다. 마지막으로 누드마우스에 인간 신경아교세포종(U87MG)을 이종이식하여 종양의 부피와 무게를 조사한 결과, CMGe열수 추출액 처리군에서 종양 부피와 무게의 감소를 확인할 수 있었다. 이런 결과는 CMGe가 앞으로 의약 및 제약산업에 다양하게 적용될 수 있을 것으로 기대된다. Cordyceps militaris has been used in traditional Chinese medicine owing to its anticancer and immunomodulatory activities. Germanium compounds have also been shown to be associated with many pharmacological functions, such as antimicrobial, antiviral, antitumor, antimutagenic, and immunomodulating effects. In this study, we examined the biological properties of hot water extract from mycelial liquid culture of germanium-enriched C. militaris (CMGe). CMGe displayed a concentration-dependent antiproliferation activity against four human cancer cell lines. The antiproliferative activity of CMGe was 2-4-fold lower than that of hot water extract from mycelial liquid culture in C. militaris (CM). However, CM had a concentration-dependent cytotoxicity to human bone marrow-derived mesenchymal stem cells (MSCs). Contrastingly, CMGe did not cause any cellular damage to MSCs. MSCs cultured with CMGe displayed an increased proliferative activity with no cytotoxic effect. The oral administration of CMGe inhibited increased tumor volume and weight compared with the control group. CMGe has the potential to be used as an industrial product in medicinal foods as well as in pharmaceutical products.

다양한 암세포 주와 MSCs에 대한 Saccharin의 항증식성 평가

최정수 ( Jeong Su Choi ),박상용 ( Sang Yong Park ),양만길 ( Man Gil Yang ),이동범 ( Dong Beom Lee ),이태복 ( Tae Bok Lee ),허지혜 ( Ji Hye Heo ),이민우 ( Min Woo Lee ),김성욱 ( Suhng Wook Kim ) 대한임상검사과학회 2016 대한임상검사과학회지(KJCLS) Vol.48 No.3

Saccharin (o-benzoic sulfimide)은 1879년에 최초로 합성된 열량이 없는 인공감미료이다. 본 연구에서, 우리는 다양한 인간 암세포주와 인간 골수에서 유래한 중간엽 줄기세포에 대한 saccharin의 생물학적 활성을 실험해보고자 한다. 4가지 인간 암세포주(H460, H157, A549, SKOV3)와 쥐암세포(Raw264.7) 그리고 인간 골수 유래 중간엽 줄기세포에 대한 세포 viability assay는 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)의 변환을 기초하여 세포독성을 실험하였다. Saccharin을 처리하지 않은 세포와 대조적으로 saccharin을 처리한 세포에서 발현 양상이 달라지는 gene을 찾기 위해, 우리는 ACP를 기초로 한 DDRT-PCR을 시행하였다. 모든 실험에 사용된 세포들은 각기 다양한 saccharin 농도로(0.0, 4.8, 7.2, 9.6, 12.0, 14.4mg/mL) 48시간 동안 처리되었다. 그 결과, 48시간 동안 다양한 saccharin 농도로 처리되면서 saccharin 처리를 하지 않은 암세포보다 saccharin 처리를 한 암세포에서 대사활성을 지닌 세포의 수가 감소하는 것을 확인할 수 있었고, 이런 세포 증식의 감소는 농도가 증가함에 따라 더욱 두드러졌다. 그리고 saccharin에 대한 반응으로 MSCs에 다른 양상으로 발현이 되는 주목할만한 gene 후보군이 2% agarose gel 상에 16개 밴드로 나타났고, 7개는 발현이 증가, 9개는 발현이 감소한 gene으로 보였다. 이 후보군중 하나는 FK506 binding protein gene이다. 이 단백질이 줄기세포의 생장활성에 어떠한 역할을 하고 있는지는 명확하지 않고 saccharin의 줄기세포 증식 활성 증가에 대한 FK506 binding protein의 자세한기능은 추후 더 연구가 필요하다. Saccharin (o-benzoic sulfimide) is the first artificial and non-caloric sweetener that was first synthesized in 1879. In this study, we examined the biological activity of saccharin against various human cancer cell lines and human bone marrow-derived mesenchymal stem cells. A viability assay based on the conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was performed to test for the cytotoxicity of saccharin about the four human cancer cell lines (H460, H157, A549 and SKOV3), one murine cancer cellline (Raw264.7), and MSCs. In order to find the differentially expressed gene in saccharin-treated MSCs against untreated MSCs, we performed annealing control primer (ACP)-based differential display reverse transcriptionpolymerase chain reaction (DDRT-PCR). All tested cells were treated with saccharin at various concentrations (0.0, 4.8, 7.2, 9.6, 12.0, 14.4 mg/mL) for 48 hr. The number of metabolically active cancer cells decreased when treated with the saccharin at various concentrations for 48 hr as compared with the untreated cells. The decrease in cell survival was more evident with increasing concentrations of saccharin. Moreover, novel candidate genes, which were differentially expressed in MSCs in response to saccharin, were identified in 16 bands on 2% agarose gel. This revealed 16. 7 up-regulated and 9 down-regulated.differentially expressed genes indicated by arrows. One of these candidate genes was a FK506-binding protein gene. The functional roles of FK506 binding proteins, with respect to the activities of stem cell proliferation, were not characterized. Further studies are required to get a better understanding of FK506-binding proteins in its roles in increasing stem cell proliferative activities from using saccharin.