• KSBB Journal
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• 제11권2호
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• pp.140-150
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• 1996

가정용 혈중 cholesterol 측정시스템을 개발하고자, 그 성능을 조절할 수 었는 벤수들의 최적화가 수 행되었다. 시스템의 주요 구성성분은 분석에 요구되는 효소들 (cholesterol esterase, cholesterol oxidase, 그리고 horseradish peroxidase) 이 부동화된 nitrocellulose membrane strip과 비 이온세척제 (Tri­t ton X-1OO) 및 발색물질 (3,3'-diaminobenzidine) 이 포함된 시료운반용액이다. 시료와 운반용액이 훈 합된 수용액을 membrane의 하단으로부터 흡수시키 면 모세관현상에 의해 시료는 부통화된 효소층으로 전달되어 연속효소반응이 일어난다. 마지막 효소반 응에서 발색물질은 산화되고 그에 따라 cholesterol 농도에 비례한 특정색이 신호로써 발생된다. 그 신호세기에 대해 효소들의 부동화조건과 운반용액의 화학조성이 최적화되었다. 최적조건 하에서 구성된 측정시스템의 농도응탑곡선은 혈중 cholesterol 농도를 측정하기에 충분히 높은 민감도를 나타내었다.

• 대한의생명과학회지
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• 제18권4호
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• pp.438-444
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• 2012

N-acylethanolamines (NAEs) including endocannabinoids, anadamide, are long chain fatty acid ethanolamines and express ubiquitously in animal and plant tissues. NAEs have several pharmacological effects including anti-inflammatory, analgesic and anorexic effects. The levels of NAEs in tissues are strictly regulated by synthesizing and hydrolyzing enzymes because NAEs are not stored in the cell but rather made on demand. NAEs are hydrolyzed to free fatty acids and ethanolamines by fatty acid amide hydrolase and N-acylethanolamine-hydrolyzing acid amidase (NAAA). Here, we suggest the fluorescence-based assay system for NAAA. We developed N-(4-methy-2-oxo-2H-chromen-7-yl)palmitamide (PAAC) as a fluorogenic substrate for NAAA and we also generated NAAA stably expressing COSM6 cell line. When extracts of cells expressing NAAA were incubated with PAAC, NAAA specifically hydrolyzed PAAC to palmitic acids and fluorogenic dye, coumarin. Release of coumarin was monitored by using fluorometer. NAAA hydrolyzed PAAC with an apparent Km of $20.05{\mu}M$ and Vmax of 32.18 pmol/mg protein/min. This assay system can be used to develop inhibitors or activators of NAAA.

• Bulletin of the Korean Chemical Society
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• 제25권1호
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• pp.115-118
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• 2004

We have examined the feasibility of using the specific interaction between mistletoe lectin I (ML I) and ${\beta}$-Dgalactose instead of the anti-ML I antibody in developing a homogeneous type competitive binding assay for ML I. We also have examined the feasibility of adapting the biotin/avidin mediated homogeneous assay for this system. Alkaline phosphatase (AKP) was employed as a single substrate enzyme label. The dose-response curve shows a detection range of 1-25 ${\mu}$g/mL and a linear response with a correlation coefficient of 0.99. To demonstrate the analytical utility of this method, 10 ${\mu}$g/mL of ML I was spiked into distilled water. The results show that the mean recovery was 10.03 ${\mu}$g/mL with an SD of 0.18. The difference between the spiked value and the mean recovery was 0.03 ${\mu}$g/mL, with a relative error of 0.3 and 1.6 % of RSD.

• Toxicological Research
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• 제16권1호
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• pp.33-37
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• 2000

Hershberger assay is known as one of the in vivo-short-term scrrning assays for endocrine disrupting chemicals (EDCs), but this method is not a validated test system. In the present study, the establishment of Hershberger assay to detect EDCs was tried using a model substance, di(n-butyl)phthalate (DBP), a plasticizer for plastics. Thirty-six immature male rats were randomly assigned to six groups: DBP 0, 40, 200, and 1000mg/kg, a positive control (flutamide 20 mg/kg), and a combination group(DBP 1000mg/kg and testosterone 50 ug/kg). DBP and flutamide were administered by gavage to male rats from day 21 to 40 post partum. Testosterone was subcutaneously injected during the same period. We evaluated body weigth gain, weights of ventral prostate, seminal vesicle, and levator ani and bulvocavernous muscle, and serum concentrations of testosterone and lutenizing hormone in male rats. The weights of seminal vesicle and levator ani and bulvocavernous muscle of males receiving 1000mg/kg of DBP was significantly lower than controls. There was no effect of DBP-treatment on body weight gain, prostate weight, and hormone concentrations. In the positive control group, the weights of seminal vesicle and levator ani and bulvocavernous muscle of males receiving 20mg/kg of flutamide were significantly lower than controls. In the combination group, there was no effect of co-treatment of DBP and testosterone on all parameters effect against DBP. This method was found to be a useful short-term screening assay system for EDCs.

• 한국해양환경ㆍ에너지학회지
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• 제10권2호
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• pp.86-92
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• 2007

본 연구는 E-screen assay를 이용해서 광양만 연안해역 퇴적물의 에스트로겐 활성분포를 정량적으로 평가하고 화학분석 결과와 비교 검토하고자 하였다. 가장 높은 에스트로겐 활성을 보인 지점은 산업단지와 섬진강 유입구에 인접한 G6, G8 이었다. E-screen assay로 얻어진 이러한 결과는 다성분 동시분석 결과와도 일치하였다. 특히 GY6, GY8 지점의 RPE는 90% 이상으로 강한 에스트로겐 작용을 나타내어 full agonist 지점으로 확인되었다. 또한 이들 지점은 EEQ가 각각 35.6 ng/g, 14.6 ng/g로 타 지점에 비해 낮아 에스트로겐 활성 효능이 상대적으로 높을 것으로 판단된다. 이러한 결과로부터, 산업단지와 섬진강 유역에 인접한 지점들을 광양만 연안 해역의 내분비계 교란 물질의 유입원으로 추정할 수 있다. 한편, 에스트로겐 활성도와 화학오염물질과의 비교결과는 상관성이 없는 것으로 나타났다.

• 한국식품영양과학회지
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• 제20권3호
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• pp.260-265
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• 1991

Salmonella/microsome assay system을 이용하여 N -nitrosodimethylamine (NDMA)의 돌연변이유발성을 검토하는 효과적인 방법과 비타민 C가 NDMA자체의 돌연변이유발과 nitrite와 2급아민으로부터의 NDMA 생성에 미치는 영향을 연구하였다. NDMA를 활성화시키기 위한 S9중 Aroclor1254로 induce시킨 hamster S9이 가장 효과가 있었으며 Aroclor 1254나 phenobarbital로 induce시킨 rat 59 mix에 의하여서는 활성화가 약하였다. DMSO와 ethanol에 NDMA를 녹였을 때는 돌연변이유발실험에서 저해효과가 나타났으나 phosphate buffer(pH 7. 4), 증류수, 95% methanol, Tween 80 + water(l : 4)는 저해 효과가 없었다. 비타민 C는 이미 생성환 NDMA에 대해서는 돌연변이 유발저해 효과를 나타내지 않았지만 nitrite와 2급 아민(dimethylamine)으로 부터의 NDMA 생성은 크게 저해하였다. 반응물에 비타민C를 첨가한 경우 Salmonella typhimurium TAl00의 revertant 수가 spontaneous숫자 수준으로 감소되었으며 HPLC를 이용한 NDMA의 분석에서도 거의 95% 까지의 정량적인 감소를 관찰할 수 있었다.

• Toxicological Research
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• 제23권3호
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• pp.263-269
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• 2007

Although taurine (2-aminoethanesulfonic acid) can inhibit oxidative stress in both animal and epidemiological studies, it is obscure whether taurine directly scavenges oxy-radicals or indirectly regulates oxidant production and/or antioxidant defense system. The reason for this discrepancy remains unknown but may be due, in part, to the lack of a validated assay system for evaluating oxy-radical scavenging capacity. The antioxidant activities of taurine and hypotaurine (2-aminoethanesulfinic acid), a precursor of taurine, against peroxyl radicals, hydroxyl radicals and peroxynitrites were determined by the total oxy-radical scavenging capacity (TOSC) assay and cell-based assay using H4IIE cells. tert-Butylhydroperoxide or hydrogen peroxide-induced cell toxicity determined by MTT assay was markedly inhibited by 10mM taurine or hypotaurine. The tert-butylhydroperoxide- or hydrogen peroxide-induced changes in oxidative stress markers, such as cellular glutathione and malondialdehyde, were ameliorated by 10mM taurine or hypotaurine. However, specific TOSC values calculated from the slope of the linear regression for taurine against peroxyl radicals, hydroxyl radicals or peroxynitrites were all less than 1 TOSC/mM. On the other hand specific TOSC values for hypotaurine against peroxyl radicals, hydroxyl radicals or peroxynitrites were 48, 2096, or 69 TOSC/mM, respectively. These results suggest that taurine protects cells against oxidative insults, which is not ascribed to directly scavenging activity of taurine against oxy-radicals. These results support the idea that the oxidation state of sulfur in antioxidants may be a determinant of oxy-radical scavenging capacity.

• Korean Journal of Acupuncture
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• 제28권2호
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• pp.23-36
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• 2011

목적 : 이 연구는 관절 및 심혈관계 질환 치료에 사용되는 송절(松節)(Pinus densiflora Gnarl)을 약침용 시료로 조제하여 본 약물의 항산화 효능을 규명하고자 하였으며 이를 다양한 시스템에서 검토하였다. 방법 : $FeCl_2$-ascorbic acid system에서 흰쥐 간조직의 지질과산화 반응을 관찰하였고, Fenton reaction system에서 자유기에 의한 plasmid DNA 분절을 유도하였다. 또한 deoxyribose assay를 통해 hydroxyl radical 소거능을 관찰하였고, NBT reduction assay로 superoxide radical 소거능을 검토하였다. 또한 human low-density lipoprotein(LDL)의 산화를 유도하기 위해 $CuSO_4$와 AAPH를 사용하였으며 relative electrophoretic mobility (REM) assay로 LDL 산화 억제 효능을 대조 항산화물질과 비교 검토하였다. 결과 : 송절 약침액은 자유기에 의한 간조직의 지질과산화(p < 0.01)및 DNA 분절을 현저하게 억제하였으며, hydroxyl radical, superoxide radical (p < 0.01), nitric oxide 및 peroxynitrite를 강하게 소거하였다. 또한 $CuSO_4$ ($IC_{50}=9.2{\pm}0.2\;{\mu}g/ml$)와 AAPH ($IC_{50}=34.8{\pm}5.1\;{\mu}g/ml$)에 의해 유도된 human LDL의 산화를 억제하였고, REM assay에서도 산화 억제 효능을 재확인할 수 있었다. 결론 : 송절 약침액은 활성산소종 및 활성질소종를 소거하였고, 지질과산화를 억제하였으며, 특히 human LDL의 산화적 손상을 방어하였다. 이에 본 약물은 자유기에 의한 심혈관의 산화적 손상을 효과적으로 보호할 것으로 판단된다.

• Toxicological Research
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• 제24권1호
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• pp.37-44
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• 2008

The in vitro cell transformation assays (CTA) were performed using BALB/3T3 murine fibroblasts and HaCaT human keratinocytes in order to evaluate concordance between both in vitro CTAs and carcinogenicity with compounds differing in their genotoxic and carcinogenic potential. Six test articles were evaluated, two each from three classes of compounds: genotoxic carcinogens (2-amino-5-nitrophenol and 4-nitroquinoline-N-oxide), genotoxic noncarcinogens (8-hydroxyquinoline and benzyl alcohol), and nongenotoxic carcinogens (methyl carbamate and N-nitrosodiphenylamine). Any foci of size $\geq$2 mm regardless of invasiveness and piling was scored as positive in CTA with BALB/3T3. As expected, four carcinogens regardless of their genotoxicity had positive outcomes in two-stage CTA using BALB/3T3 cells. However, of the two genotoxic noncarcinogens, benzyl alcohol was positive CTA finding. We concluded that, of the 6 chemicals tested, the sensitivity for BALB/3T3 system was reasonably high, being 100%. The respective specificity for BALB/3T3 assay was 50%. We also investigated the correlation between results of BALB/3T3 assay and results from HaCaT assay in order to develop a reliable human cell transformation assay. However, evaluation of staining at later time points beyond the confluency stage did not yield further assessable data because most of HaCaT cells were detached after $2{\sim}3$ days of confluency. Thus, after test article treatment, HaCaT cells were split before massive cell death began. In this modified protocol for this HaCaT system, growing attached colonies were counted instead of transformed foci 3 weeks since last subculture. Compared to BALB/3T3 assay, HaCaT assay showed moderate low sensitivity and high specificity. Despite these differences in specificity and sensitivity, both cell systems did exhibit same good concordance between in vitro CTA and rodent carcinogenicity findings (overall 83% concordant results). At present the major weakness of these in vitro CTA is lack of validation for regulatory acceptance and use. Thus, more controlled studies will be needed in order to be better able to assess and quantitatively estimate in vitro CTA data.

• 한국식품위생안전성학회지
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• 제9권1호
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• pp.23-30
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• 1994

When three systems commonly used for screening(washed platelets suspended in uffer containing Ca2+, washed platelets in buffer without Ca2+and platelet rich plasma) are compared by studying the anti-aggregating capacity of garlic extract, platelet rich plasma was the least sensitive system. The most sensitive assay system, based on the IC50s of garlic extract and 2 other platelet aggregation-inhibiting agents(vitamin K3 and propranolol), was the platelet preparation without Ca2+in the suspension buffer. This system was confirmed as the most sensitive during subsequent investigation of garlic extract's capacity to inhibit platelet ATP release. These results suggest that applying the system with washed platelet without Ca2+is most effective to screen for the anti-affregating capacity of natural food.