牛乳中GSH-Px、GST及其热稳定性研究

用去蛋白和不去蛋白两种处理方法对8头成年黑白花乳牛的乳中GSH-Px和GST的活性进行了测定;并研究了这两种酶的热稳定性。结果表明:新鲜乳和75℃消毒15s、98℃煮沸1min后的乳中GSH-Px的活性分别为118.56±97.08、25.67±23.19,0(GSH-Px活力单位);GST的活性分别为52.71±20.46、25.85±18.59,15.76±6.58(U/mL)。GST有更高的耐热性;去蛋白和不去蛋白两种处理方法对两种酶活性无显著影响(P>0.05)。     

条斑紫菜谷胱甘肽S-转移酶基因的克隆与表达分析

谷胱甘肽S-转移酶(glutathione S-transferase,GST)是一类多功能蛋白家族,主要参与解毒和抗氧化防御过程。为了研究GST在条斑紫菜叶状体解毒过程中的作用,克隆并分析了条斑紫菜一个可溶性谷胱甘肽S转移酶基因(命名为PyGST)的基因组DNA序列和cDNA序列,采用实时荧光定量PCR研究了其在铅胁迫下的表达规律。PyGST包含一个长624 bp的完整开放阅读框,编码区内含有一个长248 bp的内含子。PyGST具有GST蛋白家族的保守碱基和保守结构域。PyGST与藻类GST的亲缘关系最近,与动物Sigma型GST的亲缘关系次之;在进化树上PyGST等大多数藻类GST与动物Sigma型GST聚为一簇,表明PyGST属类Sigma型GST。铅胁迫能显著诱导PyGST表达,说明在叶状体细胞内PyGST参与了重金属铅的解毒过程。     

Resistance in the highly DDT-resistant 91-R strain of Drosophila melanogaster involves decreased penetration,increased metabolism,and direct excretion

Resistance to 4,4′-dichlorodiphenyltrichloroethane (DDT) in the 91-R strain of Drosophila melanogaster is extremely high compared to the susceptible Canton-S strain (>1500 times). In addition to enhanced oxidative detoxification, the 91-R strain also has a reduced rate of DDT penetration, increased levels of reductive and conjugative metabolism, and substantially more excretion than the Canton-S strain. Contact penetration of DDT was ∼30% less with 91-R flies, which also had significantly more cuticular hydrocarbons and a thicker, more laminated cuticle compared to Canton-S flies, possibly resulting in penetration differences. DDT was metabolized ∼1.6-fold more extensively by 91-R than Canton-S flies, resulting in dichlorodiphenyldichloroethane (DDD), two unidentified metabolites and polar conjugates being formed in significantly greater amounts. 91-R flies also excreted ∼4-fold more DDT and metabolites than Canton-S flies. Verapamil pretreatment reduced the LD₅₀ value for 91-R flies topically dosed with DDT by a factor of 10-fold, indicating that the increased excretion may involve, in part, ATP-binding cassette (ABC) transporters. In summary, DDT resistance in 91-R is polyfactorial and includes reduced penetration, increased detoxification and direct excretion.     

氧化损伤与遗传毒性的检测研究概况

概述了氧化损伤与遗传毒性的分子毒理学评价手段。简单介绍和讨论了活性氧自由基(ROS)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、过氧化物酶(POD)、谷胱甘肽解毒酶(GST)、丙二醛含量(MDA)、DNA损伤等生物学指标在毒理学领域的研究现状,重点探讨了彗星实验技术(SCGE)在检测遗传毒性方面的应用情况。     

拟南芥IAA7的原核表达及稳定性分析

以p GEX–KG为基本骨架,构建了拟南芥IAA7蛋白的原核表达载体,用IPTG诱导IAA7在3种大肠杆菌表达菌株Rosetta、BL21和Tuner中表达,利用GST SefiroseTM resin亲和树脂分离纯化GST–IAA7融合蛋白,并分析重组蛋白在体外保存时的稳定性。结果表明:GST–IAA7融合蛋白在Rosetta菌株中于25℃和0.4 mmol/L IPTG诱导下表达较好;蛋白酶抑制剂苯甲基磺酰氟(PMSF)可显著延长GST–IAA7在体外保存的时间,最后利用凝血酶切除GST标签后获得了纯化的IAA7蛋白质。     

棉铃虫核型多角体病毒感染对宿主昆虫GST活性及其表达水平的影响

【目的】研究棉铃虫核型多角体病毒(HearNPV)感染宿主昆虫后对宿主谷胱甘肽S-转移酶(GST)活性与基因表达水平的影响,明确病毒在侵染宿主过程中对宿主昆虫GST的调控作用。【方法】采用50和100PIB/只2种剂量的HearNPV病毒感染3龄棉铃虫,测定感染后不同时间试虫中肠GST活性与其编码基因的表达水平,对比分析病毒感染与未感染健康试虫的GST活性及其编码基因表达水平的差异。【结果】棉铃虫在HearNPV感染初期,GST活性显著提高,同时GST表达水平显著上调;随着感染时间的推移,GST活性与其编码基因的表达水平均显著下降,表明病毒感染后对宿主昆虫GST活性的影响与其对GST表达水平的调控相关。【结论】HearNPV感染棉铃虫过程中,病毒入侵能够激活宿主GST基因的表达,但病毒的持续感染最终会抑制GST编码基因的表达水平。     

Biochemical and molecular mechanisms of diafenthiuron resistance in the whitefly,Bemisia tabaci

B-biotype Bemisia tabaci has developed high levels of resistance to many insecticides. To investigate the risks and explore possible mechanisms of resistance to diafenthiuron in B. tabaci, a 32.8-fold diafenthiuron-resistant strain (R-DfWf) was established after selection for 36 generations compared with the susceptible strain (S-Lab). Biochemical assays showed that the activity of cytochrome P450 towards p-NA was significantly higher (4.37-fold higher) in the R-DfWf strain than in the S-Lab strain. Similarly, the carboxylesterase (COE) activity and glutathione S-transferase (GST) activity were also significantly higher (3.12- and 1.83-fold higher, respectively) in the R-DfWf strain than in the S-Lab strain. The expression of five of seven P450 genes was significantly higher (>3-fold) in the R-DfWf strain than in the S-Lab strain. The expression of COE2 was significantly higher (>2.5-fold) in the R-DfWf than in the S-Lab strain. The expression of GST and GST2 was significantly higher (>2.3-fold) in the R-DfWf than in the S-Lab. Thus, cytochrome P450, COE and GST may appear to be responsible for the resistance to diafenthiuron in B. tabaci. It is also valuable for usage of insecticides for resistance management and control of this species.     

A plant-specific tau class glutathione S-transferase from Oryza sativa with very high activity against 1-chloro-2,4-dinitrobenzene and chloroacetanilide herbicides

A plant-specific tau class GST gene homolog was successfully cloned from an Oryza sativa cDNA library by PCR using oligonucleotide primers based on the OsGSTU4 (GenBank Accession No. AF309378) sequence. The cDNA was composed of a 720-bp open reading frame encoding 239 amino acids. The deduced amino acid sequence of this gene shared over 65% sequence identity with the sequences of the tau class TaGST28e45 and ZmGST42. Conversely, the OsGSTU4 sequence showed very low identity to the GST sequences of phi, theta and zeta classes. This gene was expressed in Escherichia coli with the pET vector system, and the gene product was purified to homogeneity using GSH-Sepharose affinity column chromatography. The expressed OsGSTU4 formed a homodimer with subunits of approximately 25.5 kDa. OsGSTU4 displayed very high activity toward 1-chloro-2,4-dinitrobenzene. The activity of the OsGSTU4 was significantly inhibited by S-hexylglutathione and hematin. Plant OsGSTU4 had a unique herbicide specificity and played an important role in the detoxification reaction against fluorodifen and chloroacetanilide herbicides.     

Arsenic-induced responses in freshwater teleosts

The environment is currently polluted by thousands of chemicals or xenobiotics introduced into the environment by man to meet the demands of the modern era. Every day we encounter this negative side of human civilization, but have done little to lessen the rate of pollution. Although the entire biosphere is polluted it is water resources that are the most polluted because water is the ultimate sink for many contaminants. Thus, fish are the most vulnerable of all animal species. They are helpless because they cannot avoid the polluted habitat and face this contamination by default. Nevertheless, fish are found to survive under extreme conditions when their natural habitat has been compromised to a great extent. However, fish are highly sensitive to small environmental changes and their populations gradually dwindle if pollution continues unabated. However, we know that there are instances when water is cleaned and the rate of repopulation by different fish species has gained momentum, restoring the ecological balance. Thus, fish are considered reliable bioindicators of water pollution and fish ecotoxicology has received much attention in recent years, and fish toxicology has been able to defend a significant position in the arena of xenobiotics research over the years. This review deals with some of the major intoxication and detoxication signals manifested by fish exposed to arsenic (As), which is presently one of the most worrying metalloids in water pollution.