• 약학회지
• /
• 제38권1호
• /
• pp.12-19
• /
• 1994

Capsaicin(8-methyl-N-vanillyl-6-nonenamide) is the principal pungent component of Capsicum fruits. This work is directed to the capsaicin-hydrolyzing enzyme playing a key role in the rate limiting and critical step of capsaicin metabolism. In order to get precise information on the enzyme's subcellular location, rat liver homogenate was divided into six subcellular fractions by differential centrifugation technique: crude nuclear pellet, PNS(post nuclear supernatant) fraction, lysosomal pellet, cytosol, Tris wash fraction, micrisomes. Capsaicin-hydrolysing enzyme activity was analysed by high performance liquid chromatography(HPLC). This enzyme was found at the highest specific activity in the microsomal fraction and co-distributed with marker enzymes of the endoplasmic reticulum, NADPH-cytochrome c reductase and nucleoside diphosphatase. This is compatible with the result of ninhydrin color reaction of vanillylamine, primary metabolite of capsaicin hydrolysis, on thin layer chromatography(TLC). This enzyme is most active at pH $8.0{\sim}9.0$. Definite subcellular location of this enzyme will make it easy to proceed with further study.

• Journal of Microbiology and Biotechnology
• /
• 제14권3호
• /
• pp.612-619
• /
• 2004

A competitive direct enzyme-linked immunosorbent assay (cdELISA) was developed for selective and rapid detection of a glycopeptide antibiotic, teicoplanin (TP). TP was conjugated to bovine serum albumin (BSA) for use as an immunogen. Repeated subcutaneous injections of 0.5 mg of the conjugate was effective in generating specific polyclonal antibody (PAb) toward TP in rabbits, as determined by cdELISA. TP-horseradish peroxidase conjugate (TP-HRP) was used as an enzyme marker. The cdELISA was developed based on a competition reaction between TP-BSA PAb and TP-HRP conjugate. The TP-BSA PAb was highly sensitive (detection limit, 0.3 ng/ml and specific toward teicoplanin, showing no cross-reactivity to other glycopeptide antibiotics including vancomycin. There were good correlations ($r^2$=0.84 and 0.76, respectively) between cdELISA and microbiological assay, and high-performance liquid chromatography. The cdELISA system developed in this work is expected to be useful not only for selective and rapid monitoring of TP but also for study of TP pharmacokinetics.

• 한국식품위생안전성학회지
• /
• 제6권3호
• /
• pp.111-117
• /
• 1991

가축사료중 zearalenone 분석을 위한 enzyme linked immunosorbent assay(ELISA) 법의 개발을 위해 우선 zearalenone의 항원성을 증폭시키기 위해 zearalenone oxime 유도체를 합성한 다음 bovine serum albumin(BSA)와 conjugate를 만들고, 이를 항원으로 토끼에 면역시켜 11주에 zearalenone에 특이한 항체를 얻어냈다. 생성된 항체를 zearalenone외에 ${\alpha}-zearalenol$과는 강한 cross reactivity를 나타내었고 ${\beta}-zearalenol,\;{\alpha}-zearalenol\;및\;{\beta}-zearalenol$과는 약간의 반응을 보였으며 확립된 ELISA 조건은 당므과 같다. 먼저 시료를 methanol-phosphate buffered saline-dimethyl formate(70 : 29: 1)을 4배 첨가하여 blending 한 다음 Whatman No. 4를 통한 여액을 ELISA시료로 사용하였다. 효소 반응시간과 발색시간은 각각 $37^{\circ}C$에서 30분과 15분이었고, 흡광도는 410nm에서 ELISA reader로서 측정하였으며, 측정한계는 1~100 ppb로 매우 낮았다. 확립된 ELISA 조건으로 실제시료의 zearalenone오염도는 측정결과 24개 시료 중 4개의 시료가 양성반응을 보였고 그 함량범위는 $3.93~7.43\;\mu\textrm{g}/kg$이었다.

• Biomolecules & Therapeutics
• /
• 제15권4호
• /
• pp.261-265
• /
• 2007

Hydroxyproline (HYP) is a post-translational product of proline hydroxylation catalyzed by an enzyme prolyl 4-hydroxylase (P4H) which plays a crucial role in the synthesis of all collagens. Considering the role of collagen and its significance in many clinically important diseases such as liver fibrosis, a great deal of attention has been directed toward the development of an assay at cell-based system. The reason is that cell-based assay system is more efficient than enzyme-based in vitro system and takes much less time than in vivo system. Several assay procedures developed for P4H are laborious, time-consuming and not feasible for the massive-screening. Here, we report the cell-based assay method of prolyl 4-hydroxylase in immortalized rat hepatic stellate HSC-T6 cells. To optimize the cell culture condition to assay for HYP content, various concentrations of reagents were treated for different times in HSC-T6 cells. Our data showed that the treatment with ascorbate in a hypoxic condition for 24 h resulted in the maximal increase of HYP by 1.8 fold. Alternatively, cobalt chloride ($5\;{\mu}M$) and ascorbate ($50\;{\mu}M$) in normoxic states exhibited similar effect on the production of HYP as in a hypoxic condition. Therefore, cobalt chloride can be substituted for a hypoxic condition when an anaerobic chamber is not available. Rosiglitazone and HOE077, known as inhibitors of collagen, synthesis decreased P4H enzyme activity by 32.3% and 15%, respectively, which coincided with previous reports from liver tissues. The level of the smooth muscle ${\alpha}$-actin, a marker of activated stellate cells, was significantly increased under hypoxia, suggesting that our experimental condition could work for screening the anti-fibrotic compounds. The assay procedure took only 3 days after treatment with agents, while assays from the primary stellate cells or liver tissues have taken several weeks. Considering the time and expenses, this assay method could be useful to screen the compounds for the inhibitor of prolyl 4-hydroxylase.

• 식물조직배양학회지
• /
• 제28권3호
• /
• pp.165-171
• /
• 2001

Adenosine deaminase 유전자를 연초의 형질전환용 표지유전자로 활용할 때 형질전환체 여부를 매우 빠르고 눈으로 직접 색깔을 확인할 수 있는 새로운 방법이 개발되었다. ADA 효소는 독성인 adenosine 유도체를 비독성인 inosine 유도체와 암모니아로 변환시키는데, 이때 형성된 암모니아를 phenol-nitoprusside와 alkaline-hypochlorite 용액을 이용하여 청색으로 변환시켜 96 well plate상에서 1시간 내에 형질전환체 여부를 쉽게 확인할 수 있게 되었다. ADA효소의 substrate로서 9-D-arabinofuranosyl adenine, cordycepin, 2'-deoxyadenosine, adenosine and xylofuranosyl adenine이 모두 가능하였으며, substrate 용액의 최적조건은 adenosine 10 mM과 pH 7.5이었다. 특히 형질전환체는 ADA효소의 inhibitor인 deoxycoformycin이 함유되어 있는 용액 속에서는 adenosine을 inosine과 암모니아로 변환시키지 못해 색깔의 변화가 없었는데, 이는 형질전환체에서 색깔의 변화는 ADA효소의 작용 때문에 일어나는 것을 의미한다. 따라서 본 연구결과는 ADA 표지유전자가 도입된 형질전환체의 확인에 있어서 GUS gene system과 같이 눈으로 직접 확인할 수 있을 뿐만 아니라 매우 작은 크기의 형질전환체 절편으로 쉽고, 빠르면, 값싸게 확인할 수 있게 되었다.

• Molecules and Cells
• /
• 제21권3호
• /
• pp.411-417
• /
• 2006

We have developed a polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP) marker that can distinguish male-fertile (N) and male-sterile (S) cytoplasm in onions. The PCR-RFLP marker was located in a chloroplast psbA gene amplicon. Digesting the amplicons from different cytoplasm-containing varieties with the restriction enzyme MspI revealed that N-cytoplasm plants have a functional MspI site (CCGG), whereas the S-cytoplasm plants has a substitution in that site (CTGG), and thus no MspI target. The results obtained using this PCR-RFLP marker to distinguish between cytoplasmic male sterile factors in 35 onion varieties corresponded with those using a CMS-specific sequence-characterized amplified region (SCAR) marker. Moreover, the PCR-RFLP marker can identify N- ot S-cytoplasms in DNA sample mixtures in which they are in up to a 10-fold minority, indicating that use of the marker has high diagnostic precision. We also demonstrated the usefulness of the SNP detected in the psbA gene for high-throughput discrimination of CMS factors using Real-time PCR and a TaqMan probe assay.

• BMB Reports
• /
• 제36권2호
• /
• pp.173-178
• /
• 2003

Malignant melanoma is one of the most rapidly increasing cancer types, and patients with metastatic disease have a very poor prognosis. Detection of metastatic melanoma cells in circulation may aid the clinician in assessing tumor progression, metastatic potential, and response to therapy. Tyrosinase is a key enzyme in melanine biosynthesis. The gene is actively expressed in melanocytes and melanoma cells. Melan A is a differentiation antigen that is expressed in melanocytes. The presence of these molecules in blood is considered a marker for circulating melanoma cells. In this study, we analyzed the usefulness of this marker combination I evaluating the response to therapy in the blood of 30 patients with malignant melanoma. Circulating cells were detected by a reverse-transcriptase-polymerase-chain reaction. The tyrosinase expression was observed in 9 (30%) patients and Melan A in 19 (63.3%) patients before therapy. Following treatment, the tyrosinase mRNA was detected in only one patient, while Melan A transcripts were still present in 14 patients. We suggest that this molecular assay can identify circulating melanoma cells that express melanoma-associated antigens and may provide an early indication of therapy effectiveness.

• Molecular & Cellular Toxicology
• /
• 제4권3호
• /
• pp.211-217
• /
• 2008

To clarify the DNA damage from reactive oxygen species, we measured the DNA damage through Fpg/Endo III FLARE (Fragment Length Analysis with Repair Enzyme) assay and real time RT-PCR. The 80 SD rats assigned to 4 dose groups exposed to cumene vapor for 90 days. With Fpg/Endo III FLARE assay in hepatocytes, we found the OTM (Olive Tail Moment) and TL (Tail Length) significantly increased in no-enzyme treated and Fpg-treated control and 8 ppm groups with 28 days exposure. In Endo III-treated 8 ppm group, significantly increased the values with 90 days exposure. With lymphocytes, it was founded the values significantly increased in no-enzyme treated 800 ppm group in 28 and 90 days. It was significantly increased in Endo III-treated 80 ppm for 28 days and 800 ppm for 90 days. From the above findings, FLARE assay was suggested as being available as a biological marker for DNA damage induced by cumene exposure in SD rats. And we used real time RT-PCR for the OGG1 mRNA expression, it had dose-dependent biologic effects in 1 day exposure, but decrease the levels of rOGG1 mRNA. Our findings provide evidence that cumene exposure may cause suppression of rOGG1 in the rat hepatocytes or lymphocytes.

• 대한의생명과학회지
• /
• 제3권2호
• /
• pp.115-123
• /
• 1997

인간 $\alpha$-fetoprotein (AFP)은 간암, 위암, 생식기 종양 및 신경관 이상인 환자를 검사하고 진단하는데 유용한 지표로 알려져 있다. 본 연구에서는 사람의 AFP를 분리정제 하여 폴리클로날 항체를 생산하고 인간 혈장과 양수내의 AFP를 측정하기 위한 경쟁적 효소면역 분석법을 개발하고자 하였다. 친화크로마토그래피법과 SDS-polyacrylamide 전기영동법을 이 용하여 양수로부터 AFP를 분리하였다. 정제된 AFP를 토끼에 주사하여 폴리클로날 항체를 생산하였으며, 이중면역확산법과 Western blot 분석법을 사용하여 본 연구실에서 제조된 항체의 항원 특이성이 대단히 높음을 확인하였다. AFP와 항혈청을 이용하여 표준곡선을 얻었으며, 민감도는 5ng/ml이었고, 작용범위는 5~l,000ng/ml이었다. 분석내 CV는 4.5%이었고, 분석간 CV는 8.5%이었다. 따라서 이러한 결과로 보아 본 연구에서 개발된 경쟁적 효소면역분석법이 AFP를 측정하기에 적절하며, 간암 등의 기초연구에도 많은 기여를 할 것으로 생각된다.

• IMMUNE NETWORK
• /
• 제1권2호
• /
• pp.143-150
• /
• 2001

Background : Many investigators have found transforming growth factor-${\beta}1$ (TGF-${\beta}1$) to be elevated in tumors. Changes in responsiveness to TGF-${\beta}1$ have been linked to malignant transformation, tumor progression and tumor regression. Many malignant cell lines of epithelial or hematopoietic origin are refractory to the antiproliferative effects of TGF-${\beta}1$. However, a little is known about the association of TGF-${\beta}1$ with progression of malignant tumor. Methods : In this study, we measured the plasma level of TGF-${\beta}1$ in various cancer patients and evaluated the utility of plasma TGF-${\beta}1$ as a possible tumor marker. Plasma TGF-${\beta}1$ levels were measured using enzyme-linked immunosorbent assay in cancer patients and normal controls. Carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP) as tumor marker were compared with TGF-${\beta}1$ in the aspects of sensitivity and specificity. Results : The mean of plasma TGF-${\beta}1$ levels was $1.219{\pm}0.834ng/ml$ in normal controls, $5.491{\pm}3.598ng/ml$ in breast cancer, $12.670{\pm}10.386ng/ml$ in lung cancer, $5.747{\pm}3.228ng/ml$ in hepatocellular carcinoma and $10.854{\pm}7.996ng/ml$ in cervical cancer. In comparison with CEA and AFP, TGF-${\beta}1$ is more sensitive. Conclusion : We conclude that the high levels of TGF-${\beta}1$ are common in the plasma of cancer patients. These results suggest that the plasma TGF-${\beta}1$ level can be a potent tumor marker in various cancer patients.