瑞香狼毒根提取物对山楂叶螨的生物活性

采用不同的生物活性测定方法比较了瑞香狼毒(Stellera chamaejasme L.)根部4种不同溶剂提取物的杀螨活性。结果表明,瑞香狼毒根提取物对山楂叶螨(Tetranychus viennensis Zacher)有很好的触杀和内吸活性。在触杀活性测试中,石油醚提取物和氯仿提取物的杀螨活性最高;在内吸作用中,乙醇、氯仿和石油醚提取物的杀螨活性均较高,杀螨效果显著。在对石油醚提取物的不同溶剂萃取物进行生物活性追踪测定中发现,石油醚萃取物和氯仿萃取物具有较高的生物活性,浓度为0.6 g.L-1,山楂叶螨的24 h校正死亡率分别达到93.22%和79.66%。     

二斑叶螨对阿维菌素、哒螨灵和甲氰菊酯的抗性选育及其解毒酶活力变化

在室内模拟田间药剂的选择压力,用阿维菌素、哒螨灵和甲氰菊酯对二斑叶螨Tetranychuc urticae逐代处理,以选育其抗性种群。选育至12代,对阿维菌素抗性增长到6.72倍,对哒螨灵抗性增长到12.1倍,对甲氰菊酯抗性增长到19.9倍。酶抑制剂和离体酶活性的测定结果表明,阿维菌素抗性种群的多功能氧化酶和谷胱甘肽S-转移酶的活性均有所提高;二斑叶螨对哒螨灵的抗性可能与多功能氧化酶、羧酸酯酶的活性增强有关;而羧酸酯酶、多功能氧化酶和谷胱甘肽S-转移酶活性的增强可能是二斑叶螨对甲氰菊酯产生抗性的主要原因。     

旋覆花提取物对朱砂叶螨的生物活性及酶活性的影响

本文研究了旋覆花石油醚提取物对朱砂叶螨的毒力作用及其对相关酶活性的影响。结果表明：旋覆花石油醚提取物的杀螨活性较高,浓度为2mg?mL-1 时,校正死亡率达到92.05％。通过对旋覆花石油醚提取物进一步萃取、柱层析分离、薄层层析,发现最终得到的38个流分中,杀螨效果较好的是流分7,其校正死亡率为85.53%。流分7经GC/MS鉴定为羽扇豆醇,其校正死亡率达到66.46%。进一步测定羽扇豆醇对朱砂叶螨谷胱甘肽-S-转移酶、Ca2 -ATPase、过氧化物歧化酶的活性以及总蛋白含量的影响,结果表明经羽扇豆醇处理后,螨体内过氧化物歧化酶被激活,而谷胱甘肽-S-转移酶和Ca2 -ATPase均受到不同程度的抑制,蛋白总量在这个过程中没有明显的变化。上述结果表明旋覆花提取物羽扇豆醇可以有效地杀死朱砂叶螨,这为旋覆花作为新型植物源农药提供一定的理论依据。     

百里香杀螨活性成分的分离及鉴定

为了探讨百里香的杀螨活性成分,以山楂叶螨(Tetranychus viennensis)为供试对象,采用生物活性示踪法从百里香(Thymus mongolicus)乙醇提取物中分离纯化出5种活性成分,其化学结构经MS、1H-NMR、13C-NMR分析鉴定为百里香酚、香芹酚、松油烯-4醇、豆甾醇和β-谷甾醇.采用玻片浸渍法测试了5种化合物对山楂叶螨的触杀活性,结果表明,百里香酚和香芹酚对山楂叶螨有较强的触杀活性,12 h和24 h的触杀LC50值分别为0.103、0.135 mg·mL-1和0.048、0.096 mg·mL-1;松油烯-4-醇也有一定的杀螨活性,12 h和24 h的触杀LC50值分别为0.320和0.231 mg·mL-1;而两种甾醇类化合物β-谷甾醇和豆甾醇对山楂叶螨没有明显的触杀作用.分析认为百里香酚可能是百里香的主要杀螨活性成分之一.     

藜芦杀虫活性的初步研究

先后以乙醇、氯仿和正丁醇为溶剂,提取了熊耳山区植物藜芦根茎中的活性物质。得到3个提取物:乙醇提取物、氯仿萃取物和正丁醇萃取物,产率分别为2.93%、1.14%和0.58%。测定了3种溶剂萃取物对粘虫、蚜虫、朱砂叶螨和蚊幼虫的毒杀活性。结果说明:这几种提取物都具有良好的毒杀效果。氯仿提取物对3龄粘虫有很强的拒食作用;乙醇提取物对4龄蚊幼虫的杀虫活性最高;3种溶剂萃取物对蚜虫的毒杀能力大小次序为:氯仿提取物>正丁醇提取物>乙醇提取物;对朱砂叶螨的毒杀力大小为:乙醇提取物>氯仿提取物>正丁醇提取物。采用试管反应法和层析法对藜芦根茎提取物进行化学成分预试,结果显示:乙醇提取物、氯仿提取物和正丁醇提取物中均含有生物碱类活性物质。     

14种菊科植物提取物对松材线虫和淡色库蚊的光活化毒杀作用

以甲醇作溶剂,采用超声波提取法对14种菊科植物进行粗提,并采用浓度稀释法、结合紫外光光照处理,测定这些提取物对松材线虫和淡色库蚊的光活化毒性.研究结果表明,大部分提取物具有光活化活性,其中鲤肠、万寿菊活性最高,表现出明显的光活化毒性;500 μg/mL鲤肠提取物处理对淡色库蚊的光照活性比非光照活性高48.96倍,以1 000 μg/mL的浓度处理24 h可100%杀死供试的松材线虫;微甘菊、白花蒲公英、鱼眼草提取物也对松材线虫表现出光活化效应,光照处理的死亡率明显比非光照处理高.     

二斑叶螨对七种杀螨剂的抗药性测定及其机理研究

室内测定了相对敏感种群(S)和抗性种群(R)对常用7种杀螨剂的敏感性,并测定了羧酸酯酶、谷胱甘肽S-转移酶和乙酰胆碱酯酶3种酶的比活力。结果表明:二斑叶螨Tetranychus urticae Koch R种群已对甲氰菊酯和哒螨灵产生了抗性,抗性倍数分别为5.45和105.47。其中,甲氰菊酯对雌成螨的毒力最低(>3000mg/L),已远远超过田间推荐剂量,不宜继续使用。酶活测定结果表明:谷胱甘肽S-转移酶解毒活性的提高是二斑叶螨对甲氰菊酯产生抗性的原因之一;二斑叶螨对哒螨灵抗性的增强可能与羧酸酯酶有关。     

野生植物南山王粗提物对菜青虫的生物活性和光活化作用

野生植物南山王 (Celastrussp .)的根茎叶用甲醇、氯仿和石油醚提取 ,并制备干粉。浓度为 0l. 0 1g mL的粗提物对菜青虫PierisrapaeL .的非选择性拒食率达到 61 . 73 %～ 93 . 5 5 % ;用浸渍法测得根的粗提物对菜青虫的光活化比达到 2 5 . 5～ 3 4. 1。南山王根的氯仿提取物活性最高。     

不同生长时期黄花蒿提取物对朱砂叶螨的生物活性

选用不同生长时期(4、5和6月)黄花蒿的根、茎、叶,采用30℃～60℃石油醚、60℃～90℃石油醚、乙醇、丙酮和水溶剂等溶剂,用顺序和平行提取方法获得81种提取物,测定其对朱砂叶螨的生物活性,同时,将81种提取物分别稀释至5mg.ml-1,测定其对朱砂叶螨的触杀毒力。结果表明:在杀螨活性方面,黄花蒿的杀螨活性随植株的生长呈增加的趋势,总体表现为6月>5月>4月。6、5和4月黄花蒿叶的丙酮平行提取物活性最强,5mg.ml-1浓度处理48h对朱砂叶螨的校正死亡率为74%～86%,它们对朱砂叶螨的致死中浓度(LC50)分别为0.84、0.94和1.38mg.ml-1;处理浓度为5mg.ml-1时,它们的致死中时(LT50)分别为24.61、27.63和37.23h。     

单宁酸对棉铃虫谷胱甘肽S转移酶的影响

通过培养基混药法,研究了植物次生物质单宁酸对棉铃虫Helicoverpa armigera（Hübner）谷胱甘肽S-转移酶活性的影响。谷胱甘肽S-转移酶活性随棉铃虫发育期的进程而变化,在卵期最低,5龄、6龄幼虫和成虫期最高。用含0.005%单宁酸的饲料饲喂棉铃虫后,5龄和6龄幼虫的谷胱甘肽S-转移酶活性明显降低,分别为对照的59%和67%。单宁酸低剂量、短时间处理棉铃虫幼虫,可诱导中肠和脂肪体中的谷胱甘肽S-转移酶的活性增加,高剂量或低剂量长时间处理没有诱导增加作用,甚至还有抑制作用。单宁酸连续处理4代,对棉铃虫6龄幼虫中肠谷胱甘肽S 转移酶均有抑制作用,对脂肪体谷胱甘肽S-转移酶活性无明显影响或有抑制作用。单宁酸处理的第4代幼虫对溴氰菊酯的敏感度有增加的趋势,对甲基对硫磷的敏感度没有明显改变。     

Purification and characterization of the mouse mammary tumor virus protease expressed in Escherichia coli.

The mouse mammary tumor virus (MMTV) protease gene was cloned into pGEX-2T, an Escherichia coli expression vector containing the glutathione S-transferase coding region of Schistosoma japonicum. The chimeric protein was formed by fusion of the glutathione S-transferase with a hexapeptide which contains a thrombin cleavage site, followed by the MMTV protease. Affinity chromatography on a glutathione-Sepharose 4B column was used to isolate the chimeric protein. After thrombin cleavage, the glutathione S-transferase and the protease were separated by gel filtration chromatography on a Sephadex G-75 column. The overall yield of the protease purification procedure was about 1 mg of protease/liter of culture, and the specific activity was 380 pmol/min.micrograms of enzyme. Like other retroviral proteases, the MMTV enzyme was active as a dimer, showed maximum activity at pH between 4 and 6, and could be inhibited by pepstatin A and a phosphinic acid derivative HIV-1 protease inhibitor. Enzymatic characterization of this protease reveals its broad specificity, showing a clear preference for the oligopeptide substrate mimicking the cleavage site at the amino-terminal end of the capsid protein (kcat/Km = 9725.5 M-1.s-1). The chimeric protein was also an active dimer and showed a similar Km (17 microM) for such an oligopeptide, although its kcat was about 10 times smaller. Autocatalytic processing of the MMTV protease was observed after expression of clones containing the natural cleavage site, as it occurs at the amino-terminal end of the viral protease, instead of the thrombin-sensitive sequence.     

Different Mechanisms of Four Aluminum (Al)-Resistant Transgenes for Al Toxicity in Arabidopsis

We have characterized the mechanism of action of four transgenes (AtBCB [Arabidopsis blue copper-binding protein], parB [tobacco (Nicotiana tabacum) glutathione S-transferase], NtPox [tobacco peroxidase], and NtGDI1 [tobacco GDP dissociation inhibitor]) that independently Al resistance on transgenic Arabidopsis. All four transgenic lines showed lower deposition of callose after Al treatment than the Landsberg erecta ecotype of Arabidopsis, confirming that the four genes function to ameliorate Al toxicity. Influx and efflux experiments of Al ions suggested that the AtBCB gene may suppress Al absorption, whereas expression of the NtGDI1 gene promotes a release of Al in the root tip region of Arabidopsis. The total enzyme activities of glutathione S-transferases or peroxidases in transgenic lines carrying either the parB or NtPox genes were significantly higher than in the Landsberg erecta ecotype of Arabidopsis, and these enzyme activities were maintained at higher levels during Al stress. Furthermore, lipid peroxidation caused by Al stress was repressed in these two transgenic lines, suggesting that overexpression of these two genes diminishes oxidative damage caused by Al stress. Al-treated roots of transgenic plants were also stained by 4',6-diamino-2-phenylindole to monitor cell death caused by Al toxicity. The result suggested that cell death is repressed in the NtPox line. Analysis of F(1) hybrids between the four transgenic lines suggests that more resistant transgenic plants can be constructed by combinations of these four genes.     

Photoperiodic changes of the glutathione system of the brain under acute hypoxia]

The effect of acute hypoxic hypobaric hypoxia on the content of reduced glutathione and the activity of glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase and glutathione S-transferase, as well as 5'-nucleotidase in homogenates of juvenile male rats under conditions of varying photoperiodic duration: natural conditions of illumination, continuous illumination and continuous darkness were studied. Photoperiodic changes were revealed in the glutathione system of the control animals: the activity of glutathione peroxidase, glutathione reductase and glucose-6-phosphate dehydrogenase reduces under constant light, while the activity of glutathione peroxidase and glutathione S-transferase increases under conditions of constant darkness. The greatest inhibitory effect on the state of the glutathione system is brought about by constant light in case of acute hypoxia: the content of reduced glutathione decreases along with a sharp drop of the activity of glutathione S-transferase and glucose-6-phosphate dehydrogenase, observed against the background of decreased glutathione reductase activity. Permanent dark conditions eliminate partially or completely the negative effect of acute hypoxia on the glutathione system of the brain. The obtained results are indicator of a possibility of protecting role of melatonin in case of acute hypoxia.     

Cloning, expression, and one-step purification of the minimal essential domain of the light chain of botulinum neurotoxin type A

A truncated but functional form of the botulinum neurotoxin A light chain (Tyr 9-Leu 415) has been cloned into the three bacterial expression vectors, pET 28, pET 30, and PGEX-2T, and produced as fusion proteins. This 406-amino-acid light chain was expressed with 1 six-histidine tag (LC-pET28), 2 six histidine tags and a S-tag (LC-pET30), or a six-histidine tag and a glutathione S-transferase tag (LC-pGEX-2T). The three fusion proteins have been overexpressed in Escherichia coli, purified in a soluble form, and tested for protease activity. All three recombinant proteins were found to have similar enzymatic activity, comparable to the light chain purified from the whole toxin. The LC-pET30 protein was the most soluble and stable of the three fusion proteins, and it could be purified using a one-step affinity chromatography protocol. The purified protein was determined to be 98% pure as assessed by SDS-polyacrylamide gel. This protein has been crystallized and initial X-ray data show that the crystals diffract to 1.8 A.     

Methyl jasmonate elicits a differential antioxidant response in light- and dark-grown canola (Brassica napus) roots and shoots

Since jasmonates have been shown to mimic some of the plant'sresponses to stress, the effect of methyl jasmonate on antioxidant enzymes andcompounds was investigated in roots and shoots of light- and dark-grown canola(Brassica napus cv. Westar). The pattern of superoxidedismutase isoforms activity was also investigated. When enzyme activities werecalculated on a per gram of fresh weight basis, nearly all enzymes examinedshowed enhanced activity. However, when these activities were calculated basedon the amount of protein, methyl jasmonate induced an increase only insuperoxide dismutase activity in the roots of both light- and dark-grownseedlings. The ascorbate level was found to be higher in treated shoots,whereasthe glutathione level was found to be higher in treated roots. We conclude thatthe plant's antioxidant response to methyl jasmonate may be mainlydetermined by the type of tissue rather than by the light conditions. However,this last factor appeared to be involved in some antioxidant componentresponse,e.g. catalase activity and glutathione content.     

Chemomodulatory action of Aloe vera on the profiles of enzymes associated with carcinogen metabolism and antioxidant status regulation in mice.

The effect of two doses (30 microl and 60 microl/day/mice daily for 14 days) of the fresh leaf pulp extract of Aloe vera was examined on carcinogen-metabolizing phase-I and phase-II enzymes, antioxidant enzymes, glutathione content, lactate dehydrogenase and lipid peroxidation in the liver of mice. The modulatory effect of the pulp extract was also examined on extrahepatic organs (lung, kidney and forestomach) for the activities of glutathione S-transferase, DT-diophorase, superoxide dismutase and catalase. The positive control mice were treated with butylated hydroxyanisole (BHA). Significant increases in the levels of acid soluble sulfhydryl (-SH) content, NADPH-cytochrome P450 reductase, NADH-cytochrome b5 reductase, glutathione S-transferase (GST), DT-diaphorase (DTD), superoxide dismutase (SOD), catalase, glutathione peroxidase (GPX) and glutathione reductase (GR) were observed in the liver. Aloe vera significantly reduced the levels of cytochrome P450 and cytochrome b5. Thus, Aloe vera is clearly an inducer of phase-II enzyme system. Treatment with both doses of Aloe caused a decrease in malondialdehyde (MDA) formation and the activity of lactate dehydrogenase in the liver, suggesting its role in protection against prooxidant-induced membrane and cellular damage. The microsomal and cytosolic protein was significantly enhanced by Aloe vera, indicating the possibility of its involvement in the induction of protein synthesis. BHA, an antioxidant compound, provided the authenticity of our assay protocol and response of animals against modulator. The pulp extract was effective in inducing GST, DTD, SOD and catalase as measured in extrahepatic organs. Thus, besides liver, other organs (lung, kidney and forestomach) were also influenced favorably by Aloe vera in order to detoxify reactive metabolites, including chemical carcinogens and drugs.     

Efficient production of a bioactive, multiple disulfide-bonded protein using modified extracts of Escherichia coli

In this report, we demonstrate that a complex mammalian protein containing multiple disulfide bonds is successfully expressed in an E.coli-based cell-free protein synthesis system. Initially, disulfide-reducing activities in the cell extract prevented the formation of disulfide bonds. However, a simple pretreatment of the cell extract with iodoacetamide abolished the reducing activity. This extract was still active for protein synthesis even under oxidizing conditions. The use of a glutathione redox buffer coupled with the DsbC disulfide isomerase and pH optimization produced 40 microg/mL of active urokinase protease in a simple batch reaction. This result not only demonstrates efficient production of complex proteins, it also emphasizes the control and flexibility offered by the cell-free approach.     

In vitro antioxidant activity of Juglans regia L. bark extract and its protective effect on cyclophosphamide-induced urotoxicity in mice

Walnut (Juglans regia L.) bark has been claimed to possess anti-inflammatory, blood purifying, anticancer, depurative, diuretic and laxative activities. It contains several therapeutically active constituents, especially polyphenols. We studied the antioxidant potential of aqueous extract of walnut bark and its modulatory effect on cyclophosphamide (CP)-induced urotoxicity in Swiss albino male mice. Free radical-scavenging activity of extract was assessed in four in vitro assays. The phenolic and flavonolic contents of the extract were also measured. Walnut bark extract treatment (150 mg/kg p.o. x 10 days) resulted in protective restoration of decreased antioxidants in CP-treated (18 mg/kg i.p. x 10 days) animals. CP treatment caused decreases in the activities of catalase (CAT), glutathione peroxidase (GP), glutathione reductase (GR) and glutathione S-transferase (GST) and in the glutathione (GSH) content in urinary bladder and a significant concomitant increase in lipid peroxidation (LPO). Administration of extract restored all the antioxidants significantly and lowered the elevated LPO in the bladder. A correlation between radical scavenging capacities of the extract with phenolic content was observed thus justifying its antioxidant potential against oxidative stress-mediated urotoxicity in mice. Walnut is reported to possess antiproliferative activity. Its protective effect on CP-induced toxicity in bladder is a promising activity, which warrants possible clinical investigations on this medicinal plant.     

Tamarix gallica ameliorates thioacetamide-induced hepatic oxidative stress and hyperproliferative response in Wistar rats

Tamarix gallica, a hepatic stimulant and tonic, was examined for its ability to inhibit thioacetamide (TAA)-induced hepatic oxidative stress, toxicity and early tumor promotion response in male Wistar rats. TAA (6.6 mmol/kg body wt. i.p) enhanced lipid peroxidation, hydrogen peroxide content, glutathione S-transferase and xanthine oxidase with reduction in the activities of hepatic antioxidant enzymes viz., glutathione peroxidase, superoxide dismutase and caused depletion in the level of hepatic glutathione content. A marked increase in liver damage markers was also observed. TAA treatment also enhanced tumor promotion markers, ornithine decarboxylase (ODC) activity and [3H] thymidine incorporation into hepatic DNA. Pretreatment of rats orally with Tamarix gallica extract (25 and 50 mg/kg body weight) prevented TAA-promoted oxidative stress and toxicity. Prophylaxis with Tamarix gallica significantly reduced the susceptibility of the hepatic microsomal membrane for iron-ascorbate induced lipid peroxidation, H2O2 content, glutathione S-transferase and xanthine oxidase activities. There was also reversal of the elevated levels of liver marker parameters and tumor promotion markers. Our data suggests that Tamarix gallica is a potent chemopreventive agent and may suppress TAA-mediated hepatic oxidative stress, toxicity, and tumor promotion response in rats.