본 연구에서는 생마늘 추출 숙성물의 용매 분획물에 대한 항산화 효과와 acetylcholinesterase (AChE) 저해 효과에 대해서 조사하였다. 생마늘 추출 숙성물의 헥산, 클로포름, 에틸아세테이트 및 물 분획물을 통한 총 페놀성 화합물의 함량은 각각 3.70, 23.63, 31.27, 2.35 mg/g으로 나타났다. 또한, 에틸아세테이트 분획물이 ABTS radical scavenging activity, ferric reducing antioxidant power 및 linoleic acid를 활용한 지질의 자동산화 저해 효과에서 가장 높다는 것을 확인하였다. 추가적으로, PC12 신경 세포에서의 에틸아세테이트 분획물의 처리가 H2O2가 유발시키는 산화적 스트레스의 수준을 감소시켰다. 결국 생마늘 추출 숙성물의 에틸아세테이트 분획물이 농도 의존적으로 AChE를 저해한다는 것을 확인하였다. 그러므로 본 연구는 생마늘 추출 숙성물의 에틸아세테이트 분획물이 PC12 신경세포에 있어서 산화적 스트레스 수준을 감소시키고, 또한 AChE 저해제로서 활용될 수 있는 가능성을 확인하였다.

Western blot analysis using acetylcholinesterase (AChE)-specific antibody was conducted to determine whether AChE gene (Tuace) duplication actually results in overproduction of AChE in Tetranychus urticae (TuAChE). The protein quantities of TuAChE in seven field-collected mite populations were precisely correlated with the copy numbers. To investigate the effects of each mutation on AChE insensitivity and possible fitness cost, eight variants of TuAChE were in vitro expressed using the baculovirus expression system. Kinetic analysis revealed that the Ala391Thr mutation did not change kinetic properties of AChE, whereas the Gly228Ser and Phe439Trp mutations significantly increased the insensitivity to monocrotophos. Moreover, when the Gly228Ser and Phe439Trp mutations are present together, insensitivity increased over a thousand-fold, showing that both mutations confer resistance in a synergistic manner. Presence of the mutations, however, reduced catalytic efficiency of AChE considerably, suggesting an apparent fitness cost in monocrotophosresistant mites. Reconstitution of the multiple copies of AChE having different compositions of mutations revealed that the catalytic efficiencies of the six-copy and two-copy AChEs (resembling the AD and PyriF strains of mite, respectively) were still lower but comparable to that of wildtype AChE. These finding clearly suggested that multiple rounds of Tuace duplication was needed to compensate the reduced catalytic activity of AChE caused by mutations.

Two acetylcholinesterases (AChEs; BgAChE1 and BgAChE2) from Blattella germanica were functionally expressed using the baculovirus system. Kinetic analysis demonstrated that BgAChE2 had higher catalytic efficiency but lower substrate specificity than BgAChE1. Except paraoxon, BgAChE1 was generally less sensitive to inhibitors than BgAChE2. Western blot analysis using anti-BgAChE antibodies revealed that BgAChE1 was far more abundant in all examined tissues compared to BgAChE2, which is only present in the central nervous system. Both BgAChEs existed in dimeric form, covalently connected via a disulfide bridge under native conditions. Most fractions of BgAChE1 had a glycophosphatidylinositol (GPI) anchor, but a small fraction comprised a collagenlike tail. BgAChE2 appeared to have a collagen-GPI-fused tail. Based on the kinetic and molecular properties, tissue distribution and abundance, BgAChE1 was confirmed to play a major role in postsynaptic transmission.

아세틸콜린에스터라아제(AChE) 저해제에 의한 아세틸콜린 분해 억제는 알츠하이머 질병의 가장 확실한 치료 방법 중의 하나로 알려져 있다. 본 연구는 최근 웰빙 건강 소재로 각광을 받고 있는 과일과 채소로부터 새로운 AChE 저해제를 개발하여 항치매 식품이나 대체 의약품 생산에 응용하기 위해 과일과 채소로부터 AChE 저해 활성이 우수한 시료를 선별하고, AChE 저해 물질의 추출조건을 최적화하였다. AChE 저해 활성은 호두의 메탄올 추출물에서 72.6%(IC50=14.2㎍)로 가장 높았고, 호두의 AChE 저해 물질은 80% 메탄올로 40℃에서 12시간 동안 처리하였을 때 가장 많이 추출되었다.

The acetylcholinesterase (AChE) inhibition of 12 plant-derived insecticidal compounds (anethol, anisaldehyde, 3-carene, 1,8-cineol, ethyl-cinnamate, linalool, magnolol, ethyl-p-methoxycinnamate, p-methoxycinamic acid, safrol, terpinen-4-ol, and α-terpineol) towards adult mosquitoes were examined by using Ellman method. Result were compared with those of dichlorvos, a potent AChE inhibitor. 3-Carene strongly inhibited AChE (IC50, 5.78×10-4 M), although the inhibition of the compound was lower than dichlorvos (IC50, 1.45×10-4 M). These results indicate that 3-carene acts as an AChE inhibitor, although an involvement of other insecticidal mechanism(s) might not be ruled out.

Insensitive acetylcholinesterase (AChE) was determined to be basically involved in a EPN-resistant (ER) strain and ER contaminated susceptible (CS) strain of diamondback moth (DBM, Plutella xylostella L.), as estimated by the AChE inhibition assay using DDVP as a inhibitor in nondenaturing electrophoresis gel. ER strain exhibited very high insensitivity revel, high resistance ratio and two point mutations (G227A, A201S) in ace1. CS strain showed intermediate insensitivity level, low resistance ratio and a point mutation (G227A). This finding suggests that the A201S mutation along with reported G227A mutation (Baek et al, 2005) can be main player to develop the organophosphate resistance. Additionally, surveyed seven local population DBMs saturated G227A mutation and A201S mutation mixed in some population. Also A441G mutation was detected in some population, but there was no significant correlation. These results suggest that A201S mutation could be one of the good candidate for molecular diagnosis marker of resistance monitoring.

Insensitive acetylcholinesterase (AChE) was determined to be basically involved in a EPN-resistant (ER) strain of diamondback moth (DBM, Plutella xylostella L.), as estimated by the AChE inhibition assay using DDVP and thiodicarb as inhibitors in nondenaturing electrophoresis gel. AChEs were clearly separated into four different bands (a major band and three minor bands) in susceptible strain (CS) and all bands inhibited by used inhibitors almost same level, however, only two bands (a major band and a minor band) showed in that of ER strain and major band showed higher insensitivity. Moreover, ER strain showed cross resistance against used inhibitors and DDVP highly inhibited esterase in both strains. About 2kb of ace1,2 cloned and point mutations were detected in ER strain.

Molecular mechanisms of monocrotophos resistance in the two-spotted spider mite (TSSM), Tetranychus urticae Koch, were investigated. The resistant (AD) strain showed ca. 3,565-fold resistance compared to a susceptible (UD) strain. No significant differences in the esterase and gluthathion-S-transferase activities were found between two strains whereas AD showed a 1.9-fold higher mixed function oxidase activity. Acetylcholinesterase (AChE) inhibition assay revealed that the AChE from AD strain is 91-fold less sensitive to monocrotophos, suggestive of the target site insensitivity mechanism. Three point mutations (G228S, A391T and F439W/Y) in the AChE gene (tssmace) appeared to primarily contribute to the reduced sensitivity of AChE as judged by the correlation study of mutation frequency versus resistance levels (LC50) of several field populations. The resulting correlation coefficients of the G228S and F439W mutations were 0.711 and 0.300, respectively, suggesting that G228S mutation may play a more significant role in resistance. The A391T mutation, saturated in all field populations examined, appears to provide a base line resistance.

파밤나방(Spodoptera exigua (Hubner))은 야외집단과 실내집단들간에 살충제 감수성에서 큰 변이를 보였다. 조사된 모든 약제에서 야외집단은 비선발 실내집단보다 높은 약제 저항능력을 보였다. 약제 선발된 실내집단은 선발 약제에 대해 저항능력을 높게 발현시켰다. 이러한 약제 저항능력의 변이가 아세틸콜린에스테라제와 에스테라제 활력 변이와 비교 분석되었다. 약제 저항능력이 높은 집단일수록 아세틸콜린에스테라제 활력이 낮아지고 에스테라제활력은 높아졌다. 아세틸콜린에스테라제의 Km(Michaelis-Menten constant)값은 약제저항능력이 높은 집단일수록 증가하는 것으로 기질에 대한 이 효소의 친화도가 낮아졌다. 이 효소의 효소활력 변이는 같은 집단일수록 증가하는 것으로 기질에 대한 이 효소의 친화도가 낮아졌다. 이 효소의 효소활력 변이는 같은 집단 내에서 발육단계에 따라 차이를 나타내어 2, 3, 4령충보다는 5령충에서 특히 Km값이 증가하였다. 파밤나방의 에스테라제는 6.5% 비변성조건의 겔에서 21개 밴드를 보였다. 이중 E2, E7, E8, E11, E16, E7 밴드들은 약제저항능력이 높은 집단일수록 발현 빈도수가 높았다. 이상의 결과는 파밤나방의 살충제 감수성변이에 해독효소활력증가와 작용점변환이 포함됨을 나타낸다.