Human coagulation factor VIII domain-specific recombinant polypeptide expression

Su Jin Choi,Ki Jung Jang,Jeong-A Lim,Hye Sun Kim 대한혈액학회 2015 Blood Research Vol.50 No.2

BackgroundHemophilia A is caused by heterogeneous mutations in F8. Coagulation factor VIII (FVIII),the product of F8, is composed of multiple domains designated A1-A2-B-A3-C1-C2. FVIIIis known to interact with diverse proteins, and this characteristic may be important forhemostasis. However, little is known about domain-specific functions or their specificbinding partners. MethodsTo determine F8 domain-specific functions during blood coagulation, the FVIII domainsA1, A2, A3, and C were cloned from Hep3B hepatocytes. Domain-specific recombinantpolypeptides were glutathione S-transferase (GST)- or polyhistidine (His)-tagged,over-expressed in bacteria, and purified by specific affinity chromatography. ResultsRecombinant polypeptides of predicted sizes were obtained. The GST-tagged A2 polypeptideinteracted with coagulation factor IX, which is known to bind the A2 domain of activatedFVIII. ConclusionRecombinant, domain-specific polypeptides are useful tools to study the domain-specificfunctions of FVIII during the coagulation process, and they may be used for productionof domain-specific antibodies.

Human coagulation factor VIII domain-specific recombinant polypeptide expression

Choi, Su Jin,Jang, Ki Jung,Lim, Jeong-A,Kim, Hye Sun Korean Society of Hematology; Korean Society of Bl 2015 Blood Research Vol.50 No.2

<P><B>Background</B></P><P>Hemophilia A is caused by heterogeneous mutations in <I>F8</I>. Coagulation factor VIII (FVIII), the product of <I>F8</I>, is composed of multiple domains designated A1-A2-B-A3-C1-C2. FVIII is known to interact with diverse proteins, and this characteristic may be important for hemostasis. However, little is known about domain-specific functions or their specific binding partners.</P><P><B>Methods</B></P><P>To determine <I>F8</I> domain-specific functions during blood coagulation, the FVIII domains A1, A2, A3, and C were cloned from Hep3B hepatocytes. Domain-specific recombinant polypeptides were glutathione S-transferase (GST)- or polyhistidine (His)-tagged, over-expressed in bacteria, and purified by specific affinity chromatography.</P><P><B>Results</B></P><P>Recombinant polypeptides of predicted sizes were obtained. The GST-tagged A2 polypeptide interacted with coagulation factor IX, which is known to bind the A2 domain of activated FVIII.</P><P><B>Conclusion</B></P><P>Recombinant, domain-specific polypeptides are useful tools to study the domain-specific functions of FVIII during the coagulation process, and they may be used for production of domain-specific antibodies.</P>

한국인 좌심실 비대증 환자들에서 파브리병 선별검사의 의의

박형두,조성윤,이수연,전은석,박승우,이상훈,이상철,최진오,박성지,장성아,김형관,기창석,김종원,진동규,Park, Hyeong-Du,Jo, Seong-Yun,Lee, Su-Yeon,Jeon, Eun-Seok,Park, Seung-U,Lee, Sang-Hun,Lee, Sang-Cheol,Choe, Jin-O,Park, Seong-Ji,Jang, Seong-A,K 대한유전성대사질환학회 2014 대한유전성대사질환학회지 Vol.14 No.2

목적: 파브리병(Fabry disease)은 alpha-galactosidase A의 결핍으로 인하여 리소좀에 globotriaosylceramide(Gb3)가 축적되어 여러 장기에 이상을 일으키는 질병이다. 본 연구에서는 파브리병의 만성 합병증 중 심장 질환을 주로 보이는 환자들, 그 중에서도 좌심실 비대증을 보이는 한국인 환자들을 대상으로 파브리병의 빈도를 알아보고자 하였다. 방법: 좌심실비대증을 진단받은 환자 257명을 연구대상으로 선정하였고, 남성이 172명(평균 56세, 범위 30-81세), 여성이 84명(평균 66세, 범위 45-85세)이었다. 파브리병 선별을 위하여 고성능액체크로마토그래피-탠덤질량분석기를 이용하여 소변 Gb3 농도를 측정하였다. 확진은 형광분석법에 의한 말초혈액의 alpha-galactosidase A 활성도와 염기서열분석법에 의한 GLA 유전자 돌연변이 유무를 검사하여 이루어졌다. 결과: 소변 Gb3 검사에서 cutoff (25 ug/mmoL creatinine)를 초과하는 환자는 4명이었지만, 최종적으로 추가 검사를 통해 진단된 파브리병 환자는 여성 환자 한 명이었다(1/257명, 0.4%). 확진된 환자는 54.3 ug/mmoL creatinine의 Gb3 농도와 15.5 nmole/hr/mg protein (참고범위, $55.2{\pm}12.7nmole/hr/mg$ protein)의 alpha-galactosidase A 활성도를 보였다. GLA 유전자에서는 c.796G>A (p.D266N) 돌연변이가 이형접합체로 관찰되었다. 추가로 시행한 가족검사에서 환자의 딸은 아직 파브리병의 증상을 보이지 않았지만, 엄마와 같은 GLA 돌연변이(c.796G>A)를 가지고 있었으며, alpha-galactosidaseA 활성도는 42.5 nmole/hr/mg protein, 소변 Gb3 농도는 25.5 ug/mmoL creatinine을 나타냈다. 결론: 한국인 좌심실 비대증을 가진 환자들에서 파브리병의 유병율은 0.4%였다. 유병율이 낮아 보임에도 불구하고, 파브리병 진단 전 환자와 가족 구성원을 발견할 수 있는 장점 덕분에 선별검사의 의의가 있는 것으로 사료된다. Objectives: Fabry disease (FD) is a lysosomal storage disease caused by the inappropriate accumulation of globotriaosylceramide (Gb3) in tissues due to a deficiency in the enzyme ${\alpha}$-galactosidase A. Hypertrophic cardiomyopathy is one of the chronic complications of FD. We tried to evaluate the prevalence of Fabry disease in the Korean patients with left ventricular hypertrophy (LVH). Methods: A total of 257 patients with LVH were recruited and they were 172 males (mean 56 years, range 30-81 years) and 84 females (mean 66 years, range 45-85 years). Urinary Gb3 was used to screen FD by high performance liquid chromatography-tandem mass spectrometry. Confirmatory tests were done by alpha-galactosidaseA activity using fluorometric assay and by GLA mutation analysis using sequencing. Results: Four patients were screening positive by urinary Gb3 analysis (cutoff, 25 ug/mmol creatinine). But, one female patient was diagnosed with FD confirmed by enzyme analysis in leukocytes as well as by genetic analysis (1/257 patients, 0.4%). She showed 54.3 ug/mmoL creatinine of Gb3 and 15.5 nmole/hr/mg protein (reference range, $55.2{\pm}12.7nmole/hr/mg$ protein) of alphagalactosidase A activity. And she had a heterozygous GLA mutation of c.796G>A (p.D266N). Her daughter was found to be a carrier for FD confirmed by GLA mutation analysis. Asymptomatic carrier showed 25.5ug/mmol creatinine of Gb3 and 42.5 nmole/hr/mg protein (reference range, $55.2{\pm}12.7nmole/hr/mg$ protein) of alpha-galactosidase A activity. Conclusions: The prevalence of FD in Koran patients with LVH was detected as 0.4%. Although the prevalence seems to be low, screening studies are of great importance for detecting hidden cases as well as for identifying other effected family members.

Anti-inflammatory properties of neutral lipids, glycolipids, and phospholipids isolated from Ammodytes personatus eggs in LPS-stimulated RAW264.7 cells

Gyoung Su Choi,A-Yeong Jang,Weerawan Rod-In,Woo Jung Park 한국식품영양과학회 2021 한국식품영양과학회 학술대회발표집 Vol.2021 No.10

Total lipids were extracted from A. personatus (Pacific sand lance) eggs, and then they were separated into neutral lipids, glycolipids and phospholipids. Anti-inflammatory activities of three lipids including neutral lipids, glycolipids and phospholipids isolated from A. personatus eggs were determined by the production of nitric oxide (NO), the gene expression, and the protein expression in LPS-stimulated RAW264.7 murine macrophages. The cells were pre-treated with the lipids at various concentration (0.5, 1.0, 1.5, and 2.0%), and stimulated with LPS. The results demonstrated that three lipids significantly reduced the NO production and the mRNA expression of immune-associated genes, such as iNOS, COX-2, IL-1β, IL-6, and TNF-α, in dose-dependent manner. All lipids also down-regulated the protein expression of phosphorylated NF-κB-p65 and MAPKs (p38, JNK and ERK1/2) signaling pathway. These results suggest that neutral lipids, glycolipids and phospholipids isolated from A. personatus egg exerts the anti-inflammatory activities via the inhibition of NF-κB and MAPKs activation.

Significant interarm blood pressure difference predicts cardiovascular risk in hypertensive patients : CoCoNet study

Kim, Su-A,Kim, Jang Young,Park, Jeong Bae Wolters Kluwer Health 2016 Medicine Vol.95 No.24

<▼1><P>Supplemental Digital Content is available in the text</P></▼1><▼2><P><B>Abstract</B></P><P>There has been a rising interest in interarm blood pressure difference (IAD), due to its relationship with peripheral arterial disease and its possible relationship with cardiovascular disease. This study aimed to characterize hypertensive patients with a significant IAD in relation to cardiovascular risk. A total of 3699 patients (mean age, 61 ± 11 years) were prospectively enrolled in the study. Blood pressure (BP) was measured simultaneously in both arms 3 times using an automated cuff-oscillometric device. IAD was defined as the absolute difference in averaged BPs between the left and right arm, and an IAD ≥ 10 mm Hg was considered to be significant. The Framingham risk score was used to calculate the 10-year cardiovascular risk. The mean systolic IAD (sIAD) was 4.3 ± 4.1 mm Hg, and 285 (7.7%) patients showed significant sIAD. Patients with significant sIAD showed larger body mass index (<I>P</I> < 0.001), greater systolic BP (<I>P</I> = 0.050), more coronary artery disease (relative risk = 1.356, <I>P</I> = 0.034), and more cerebrovascular disease (relative risk = 1.521, <I>P</I> = 0.072). The mean 10-year cardiovascular risk was 9.3 ± 7.7%. By multiple regression, sIAD was significantly but weakly correlated with the 10-year cardiovascular risk (β = 0.135, <I>P</I> = 0.008). Patients with significant sIAD showed a higher prevalence of coronary artery disease, as well as an increase in 10-year cardiovascular risk. Therefore, accurate measurements of sIAD may serve as a simple and cost-effective tool for predicting cardiovascular risk in clinical settings.</P></▼2>

Compounds Obtained from Sida acuta with the Potential to Induce Quinone Reductase and to Inhibit 7,12-Dimethylbenz-[a]anthracene-Induced Preneoplastic Lesions in a Mouse Mammary Organ Culture Model

Jang, Dae-Sik,Park, Eun-Jung,Kang, Young-Hwa,Su, Bao-Ning,Hawthorne, Michael-E.,Vigo, Jose-Schunke,Graham, James-G.,Cabieses, Fernando,Fong, Harry H.S.,Mehta, Rajendra-G.,Pezzuto, John-M.,Kinghorn, A. The Pharmaceutical Society of Korea 2003 Archives of Pharmacal Research Vol.26 No.8

Activity-guided fractionation of the EtOAc-soluble extract of the whole plants of Sida acuta using a bioassay based on the induction of quinone reductase (OR) in cultured Hepa 1c1c7 mouse hepatoma cells, led to the isolation of ten active compounds of previously known structure, quindolinone (1), cryptolepinone (2), 11-methoxyquindoline (3), N-trans-feruloyltyramine (4), vomifoliol (5), loliolide (6), 4-ketopinoresinol (7), scopoletin (8), evofolin-A (9), and evofolin-B (10), along with five inactive compounds of known structure, ferulic acid, sinapic acid, syringic acid, ($\pm$)-syringaresinol, and vanillic acid. These isolates were identified by physical and spectral data measurement. A new derivative of quindolinone, 5,10-dimethylquindolin-11-one (1a) was synthesized and characterized spectroscopically. Of the active substances, compounds 1-3 and 1a exhibited the most potent QR activity, with observed CD (concentration required to double induction) values ranging from 0.01 to 0.12 $\mu$ g/mL. Six compounds were then evaluated in a mouse mammary organ culture assay, with cryptolepinone (2), N-trans-feruloyltyramine (4), and 5,10-dimethylquindolin-11-one (1a) found to exhibit 83.3, 75.0, and 66.7% inhibition of 7,12-dimethylbenz[a]anthracene-induced preneoplastic lesions, respectively, at a dose of 10 $\mu\textrm{g}$/mL.

Monte Carlo Study on the Wetting Behavior of a Surface Texturized with Domed Pillars

Kim, Hyojeong,Lee, Su In,Matin, Mohammad A.,Zhang, Zhengqing,Jang, Jihye,Ha, Man Yeong,Jang, Joonkyung American Chemical Society 2014 The Journal of Physical Chemistry Part C Vol.118 No.45

<P>A lattice gas Monte Carlo simulation was performed to examine the wetting properties of a surface texturized with nanometer-sized, dome-shaped pillars. The vapor and liquid phases of the gap between the pillars were related to the Wenzel and Cassie–Baxter states of a macroscopic water droplet resting on top of the pillars. We studied the effects of the pillar size by systematically varying its height from 6 to 53 nm for a fixed ratio of the height to its width. With increasing interpillar spacing or pressure, the liquid on top of the domed pillars penetrated smoothly down into the gap between the pillars. This wetting transition contrasts with that observed for the gap between rectangular or cylindrical pillars, where a liquid abruptly fills in the interpillar gap at a critical interpillar spacing or pressure. The gap between the domed pillars was more susceptible to the intrusion of the bulk liquid on top of the pillars, due to the open geometry of the gap between the domed pillars. Also, the liquid penetrating into the gap between the domed pillars was locally more fluctuating in density and compressible than that penetrating into the gap between square or cylindrical pillars. This enhanced density fluctuation however was local and did not propagate into the bulk liquid sitting on top of the pillars. Simple analytic expressions of the critical spacing and pressure at which the wetting transition occurs for the domed pillars were derived using continuum theory. These continuum results agreed reasonably well with the present molecular simulations, even for pillars as small as a few nanometers in width.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/jpccck/2014/jpccck.2014.118.issue-45/jp5076077/production/images/medium/jp-2014-076077_0013.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/jp5076077'>ACS Electronic Supporting Info</A></P>

Prevalidation trial for a novel <i>in vitro</i> eye irritation test using the reconstructed human cornea-like epithelial model, MCTT HCE™

Yang, Hyeri,Kim, Da-eun,Jang, Won-Hee,An, Susun,Cho, Sun-A,Jung, Mi-Sook,Lee, Ji Eun,Yeo, Kyung-Wook,Koh, Sang Bum,Jeong, Tae-Cheon,Kang, Mi-Jeong,Chun, Young-Jin,Lee, Su-Hyon,Lim, Kyung-Min,Bae, Seun Pergamon 2017 Toxicology in vitro Vol.39 No.-

<P><B>Abstract</B></P> <P>Here, we report the results of a prevalidation trial for an <I>in vitro</I> eye irritation test (EIT) using the reconstructed human cornea-like epithelium, MCTT HCE™. The optimal cutoff to determine irritation in the prediction model was established at 35% with the receiver operation characteristics(ROC) curve for 126 substances. Within-lab(WL) and between-lab(BL) reproducibility was tested for 20 reference substances by 3 participating laboratories. Viability data described by mean±SD or ±1/2 difference between duplicate wells, and scatter plots, demonstrated the WL/BL consistency. WL/BL concordance with the binary decision, whether non-irritant or irritant was estimated to be 85–95% and 95%, respectively. WL/BL reproducibility of viability data was further supported by a strong correlation(ICC, <I>r</I> >0.9). WL/BL agreement of binary decisions was also examined by Fleiss' Kappa statistics, which showed a strong level of agreement (>0.78), nevertheless weaker than the reproducibility of the viability. The EIT with MCTT HCE™ exhibited a sensitivity of 82.2% (60/73), a specificity of 81.1% (43/53), and an accuracy of 81.8% (103/126) for 126 reference substances (for liquids; a sensitivity of 100% (47/47), a specificity of 70.6% (24/34), and an accuracy of 87.7% (71/81), and for solids, a sensitivity of 50% (13/26), a specificity of 100% (19/19), and an accuracy of 71.1% (32/45), suggesting that the accuracy is satisfactory but the sensitivity needs improvement, which shall be addressed through correcting the poor sensitivity for solid substances in future full validation trials.</P> <P><B>Highlights</B></P> <P> <UL> <LI> MCTT™ HCE EIT employing water-soluble formazan WST-1, demonstrated a performance comparable to other RhCEs. </LI> <LI> Within- and between-lab reproducibility for 3 labs with 20 chemicals were 85–95% and 95% respectively. </LI> <LI> Predictive capacity for 126 chemicals was sensitivity 82.2%, specificity 81.1% and accuracy 81.8% </LI> </UL> </P>

의료용 대마 재배시설 내 범죄예방을 위한 서비스디자인 연구

장수아 ( Jang Su A ),반영환 ( Pan Young Hwan ) 디자인융복합학회(구.한국인포디자인학회) 2021 디자인융복합연구 Vol.20 No.3

대마 관련 범죄가 증가하는 가운데 경상북도 안동시가 경북 산업용 헴프 규제자유특구로 지정되었다. 하지만 특구의 목적과는 달리 종자를 남용하는 사례가 발생해 우려의 목소리가 제기되는 상황이다. 이에 본 연구는 의료용 대마 재배시설의 범죄 예방을 위한 서비스디자인을 개발하고자 한다. 연구에서는 현장조사(Field Research), 인터뷰(Contextual Interview), 쉐도잉(Shadowing)과 같은 서비스디자인 방법론을 활용해 의료용 대마 재배시설의 범죄 발생 3요소(주체, 내부 환경, 작물 특성)를 정의했으며, 5가지의 서비스 핵심이슈를 도출하였다. 이후, 도출된 핵심이슈를 바탕으로 아이데이션 워크숍(Ideation Workshop)을 진행하고 그 결과로 의료용 대마 재배시설의 범죄예방 3요소(구조, 시스템, 운영)와 각 요소에 따른 해결방안을 제시하였다. 본 연구는 실제 의료용 대마 재배시설과 밀접 이해관계자를 바탕으로 시행된 최초 연구라는 점에 의의가 있으며, 향후 의료용 대마 재배시설의 표준 운영방안 설계를 위한 기초연구로 활용될 것으로 기대한다. Andong City, Gyeongsangbuk-do was designated as Industrial Hemp Regulation Free Special Zone. However, There is a case of abuse of seeds, raising concerns. This study aims to develop a service design for crime prevention of medical cannabis cultivation facilities. In this study, three factors(Criminal, Internal Environment, Plant characteristics) of medical cannabis cultivation facilities were defined by using service design methodology such as Field Research, Contextual Interview, and Shadowing, and five core issues were derived. After that, based on the core issues, Ideation Workshop was conducted. As a result, Three factors of crime prevention(Structure, System, Operation) and solutions of medical cannabis cultivation facilities were defined and solutions were designed according to each factor. This study is meaningful in that it is the first research conducted based on actual medical cannabis cultivation facilities and close stakeholders. It is expected that it will be used as a basic research for designing standard operation plan of medical cannabis cultivation facility in the future.

Homeobox protein Hhex negatively regulates Treg cells by inhibiting Foxp3 expression and function

Jang, Sung Woong,Hwang, Soo Seok,Kim, Hyeong Su,Kim, Min Kyung,Lee, Woo Ho,Hwang, Soh Un,Gwak, Jinu,Yew, Si Kyoung,Flavell, Richard A.,Lee, Gap Ryol National Academy of Sciences 2019 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF Vol.116 No.51

<P><B>Significance</B></P><P>Regulatory T (Treg) cells play an essential role in maintaining immune homeostasis. Studying factors that control Treg differentiation and function are critically important to understand immune homeostasis. In this manuscript, we discovered that transcription factor Hhex exerts an inhibitory effect on Treg cell differentiation and function. Hhex-overexpressing Treg cells lose their Foxp3 expression and fail to suppress immune responses. Hhex directly binds to Foxp3 protein and the <I>Foxp3</I> locus and inhibits expression of Foxp3 and its target genes. Thus, Hhex plays an essential role in inhibiting Treg cell differentiation and function via inhibition of Foxp3. This study will benefit clinical research in developing a therapeutic strategy for Treg cell-related diseases.</P><P>Regulatory T (Treg) cells play an essential role in maintaining immune homeostasis, but the suppressive function of Treg cells can be an obstacle in the treatment of cancer and chronic infectious diseases. Here, we identified the homeobox protein Hhex as a negative regulator of Treg cells. The expression of Hhex was lower in Treg cells than in conventional T (Tconv) cells. Hhex expression was repressed in Treg cells by TGF-β/Smad3 signaling. Retroviral overexpression of Hhex inhibited the differentiation of induced Treg (iTreg) cells and the stability of thymic Treg (tTreg) cells by significantly reducing Foxp3 expression. Moreover, Hhex-overexpressing Treg cells lost their immunosuppressive activity and failed to prevent colitis in a mouse model of inflammatory bowel disease (IBD). <I>Hhex</I> expression was increased; however, <I>Foxp3</I> expression was decreased in Treg cells in a delayed-type hypersensitivity (DTH) reaction, a type I immune reaction. Hhex directly bound to the promoters of <I>Foxp3</I> and other Treg signature genes, including <I>Il2ra</I> and <I>Ctla4</I>, and repressed their transactivation. The homeodomain and N-terminal repression domain of Hhex were critical for inhibiting Foxp3 and other Treg signature genes. Thus, Hhex plays an essential role in inhibiting Treg cell differentiation and function via inhibition of Foxp3.</P>