• Toxicological Research
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• 제35권1호
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• pp.37-44
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• 2019

A major predictor of the efficacy of natural or synthetic cannabinoids is their binding affinity to the cannabinoid type I receptor ($CB_1$) in the central nervous system, as the main psychological effects of cannabinoids are achieved via binding to this receptor. Conventionally, receptor binding assays have been performed using isotopes, which are inconvenient owing to the effects of radioactivity. In the present study, the binding affinities of five cannabinoids for purified $CB_1$ were measured using a surface plasmon resonance (SPR) technique as a putative non-isotopic receptor binding assay. Results were compared with those of a radio-isotope-labeled receptor binding assay. The representative natural cannabinoid ${\Delta}^9$-tetrahydrocannabinol and four synthetic cannabinoids, JWH-015, JWH-210, RCS-4, and JWH-250, were assessed using both the SPR biosensor assay and the conventional isotopic receptor binding assay. The binding affinities of the test substances to $CB_1$ were determined to be (from highest to lowest) $9.52{\times}10^{-3}M$ (JWH-210), $6.54{\times}10^{-12}M$ (JWH-250), $1.56{\times}10^{-11}M$ (${\Delta}^9$-tetrahydrocannabinol), $2.75{\times}10^{-11}M$ (RCS-4), and $6.80{\times}10^{-11}M$ (JWH-015) using the non-isotopic method. Using the conventional isotopic receptor binding assay, the same order of affinities was observed. In conclusion, our results support the use of kinetic analysis via SPR in place of the isotopic receptor binding assay. To replace the receptor binding affinity assay with SPR techniques in routine assays, further studies for method validation will be needed in the future.

• BMB Reports
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• 제28권5호
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• pp.437-442
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• 1995

The potency of yeast-derived methionyl-free human growth hormone (rhGH), which was obtained by removal of the N-terminal Met from methionyl-hGH, was estimated by in vitro and in vivo assays. In radio-receptor assay where the binding affinity of growth hormone to the receptor was estimated, the recombinant hGH showed 2.9 international units (IU) per mg of specific activity. In contrast, pitUitary-derived human growth hormone had a slightly lower receptor binding activity (2.5 IU/mg) compared with recombinant growth hormone. For the in vivo assay, efficacy of rhGH was tested by use of hypophysectomized rats, in which pituitary organs were surgically removed, resulting in the termination of growth hormone secretion. The weight-increase in rats by the injection of rhGH was almost identical to the result obtained by the injection of the same amount of pituitary-derived (international standard) hGH. A comparision of the secondary structures of rhGH and rMet-hGH by circular dichroism spectrophotometer demonstrated that the removal of the methionyl residue from rMet-hGH did not exert any effect on the structure of the growth hormone. In conclusion, methionyl-free human growth hormone produced from yeast was highly potent in biological activity and maintained a legitimate three dimensional structure.

• BMB Reports
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• 제34권2호
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• pp.123-129
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• 2001

Cellular response to ionizing radiation is affected by cell types, radiation doses, and post-irradiation time. Based on the trypan blue dye exclusion assay in normal oral mucosal cells (OM cells), a 48 h post-irradiation was sufffcient and an adequate time point for the evaluation of radiation sensitivity Its $LD_{50}$ was approximately 1.83 Gy To investigate possible biomarkers useful for the biological radiodosimetry of normal epithelial cells (p53, c-fos, cyclin D1, cdc-2, pRb) EGF receptor phosphorylation and Erk activation were evaluated at different radiation doses and different post-irradiation times. From 0.5 Gy, p53 was accumulated in the nucleus of basal cells of the OM raft culture at 4 h post-irradiation and sustained up to 24 h post-irradiation, which suggests that radiation-induced apoptosis or damage repair was not yet completed. The number of p53 positive cells and biosynthesis of p53 were correlated with radiation doses. Both cyclin D1 and c-fos were only transiently induced within 1 h post-irradiation. Cyclin D1 was induced at all radiation doses. However, cfos induction was highest at 0.1 Gy, approximately 7.3 fold more induction than the control, whose induction was reduced in a reverse correlation with radiation dose. The phosphorylation pattern of cdc-2 and pRb were unaffected by radiation. In contrast to A431 tails overexpressing the EGF receptor approximately 8.5 fold higher than normal epithelial, the OM cells reduced the basal level of the EGF receptor phosphorylation in a radiation dose dependent fashion. In conclusion, among radiation-induced biomolecules, the p53 nuclear accumulation may be considered for the future development of a useful marker far biological radiodosimetry in normal epithelial tissue since it was sustained for a longer period and showed a dose response relationship. Specific c-fos induction at a low dose may also be an important finding in this study It needs to be studied further for the elucidation of its possible connection with the low dose radio-adaptive response.

• 핵의학기술
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• 제13권3호
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• pp.175-180
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• 2009

서론 : 갑상선 기능 항진을 보이는 환자를 대상으로 항갑상선제 치료후 재발유무를 예측하거나 정확한 그레이브스병을 진단하기 위해 임상에서는 갑상선자극호르몬 결합악제면 역글로블린을 측정하는데 지금까지 1세대 liquid phase (RIA Precipitate)를 사용하였다. 그 후 예민도가 높아진 다음 세대 측정법이 다양하게 개발되었으며 실제 임상적 유용성이 보고되었다. 따라서 세대별 갑상선자극호르몬 결합억제면역글로블린 측정법 임상적 유용성 검토해 보고자 한다. 실험재료 및 방법 : 2009년 1월에서 3월까지 본원 강남건강증진센터에 내원한 환자 중 TSH, FT4, Microsome Ab 측정값이 정상범위에 드는 30명 (남자 20명, 여자 10명)을 대조군으로 하였다. 마찬가지로 2009년 1월에서 3월까지 본원 내분비내과를 내원한 환자 중 TSH<$0.05\;{\mu}IU/mL$, FT4>1.80 ng/dL인 갑상선기능항진증 진단을 받은 58명 (남자14명, 여자 44명)을 실험군으로 하였다. 측정법은 각 세대별로 1세대에서 3세대까지 각각 2가지종류의 시약을 사용하였으며, 확진 검사로 TS-Ab 검사를 실시하였다. 결과 : 대조군을 대상으로 각 세대별 측정법의 특이도를 확인하였다. (Specificity=100%, n=30) 실험군에서 민감도는 1세대 RSR<%> (79.3%, n=58), 1세대 RSR (51.7%, n=58), 2세대 RSR-CT (93.1%, n=58), 2세대 BRAHMS-CT (98.3%, n=58), 3세대 ELISA (94.6%, n=56), 3세대 ECLIA (97.7%, n=58), TS-Ab<%> (93.5%, n=46)이었다. 결론 : 갑상선자극호르몬 결합억제면역글로블린 측정법의 세대별 검토 결과 TS-Ab(%)를 확진 검사로 기준했을 때 3세대 ECLIA에 대해 2세대 BRAHMS-CT 측정법이 가장 높은 상관성을 보였다($R^2$=0.923). 따라서 기존에 사용했던 1세대 측정법을 대신하여 2세대 측정법을 사용함으로써 보다 정확하고 신속한 결과보고에 유용할 것으로 판단된다.