Expression and characterisation of a Psoroptes ovis glutathione S-transferase

The astigmatid mite Psoroptes ovis is the causative agent of sheep scab, a highly contagious parasitic disease of sheep. Infection causes severe allergic dermatitis, resulting in damage to the fleece and hide, loss of condition and occasional mortality. Interest in the P. ovis allergens led us to characterise a glutathione S-transferase (GST) which displays homology to GST allergens isolated from the house dust mite, Dermatophagoides pteronyssinus and the cockroach, Blatella germanica. A cDNA encoding a mu-class GST from P. ovis was expressed in Escherichia coli and the recombinant protein purified for biochemical analysis. SDS-PAGE analysis indicated that the purified product was homogeneous and had an apparent molecular weight of 30 kDa. The recombinant GST (rGST) is active towards the substrate 1-chloro-2,4-dinitrobenzene (CDNB), whereas 1,2-dichloro-4-nitrobenzene (DCNB) is a poor substrate. The recombinant protein was also tested for recognition by IgE and IgG antibodies in serum from P. ovis na?ve and P. ovis infested sheep. Neither IgE nor IgG antibodies were detected to the rGST. Prausnitz--Küstner testing with rGST did not provoke a characteristic weal and flare response. Biopsies collected at the PK test sites were stained for eosinophils, neutrophils, mast cells and basophils. Neutrophil, mast cell and basophil counts were not significantly different to the controls. Eosinophil numbers were significantly higher than controls, but were not due to an IgE response.     

First report of a Rhipicephalus microplus tick population multi-resistant to acaricides and ivermectin in the Mexican tropics

We document the presence of a Rhipicephalus microplus tick population resistant to acaricides (organophosphates (OP), synthetic pyrethroids (SP), amitraz) and macrocyclic lactones (ML) (ivermectin). Engorged females of R. microplus were collected from a cattle farm in Veracruz, Mexico, to evaluate acaricide and ivermectin resistance. The modified larval packet test (LPT) was used to detect OP (chlorpiriphos and diazinon) and SP (flumethrin, deltamethrin and cypermethrin) resistance and the larval immersion test (LIT) to detect resistance to amitraz and ivermectin. Both, LPT and LIT were performed twice at different times with different collected samples. Mortality data with ivermectin were subjected to probit analysis to obtain lethal concentrations and resistance ratios (RR) using an ivermectin-susceptible strain (Deutch) as a reference. The R. microplus population showed resistance to all acaricides tested, with different mortalities at the discriminate dose: chlorpiriphos (1%), diazinon (24.2%), flumethrin (92.8%), deltamethrin (94.2%), cypermethrin (98.0%) and amitraz (1.5%). The studied tick population also showed resistance to ivermectin with a resistance ratio at 99% of 9.58 and 6.52 in the first and second evaluation, respectively. We report for the first time a R. microplus population in Mexico with different levels of resistance to OP, SP, amidines (Am) and ivermectin. The uncontrolled use of these products in the study area may promote the complete failure of tick control within a short period of time.     

Acaricidal activity of aqueous extract and synthesized silver nanoparticles from Manilkara zapota against Rhipicephalus (Boophilus) microplus

Traditional parasite control is primarily based on the use of chemical acaricides, which unfortunately have many negative side effects. The aim of the present study was to evaluate the effect of plant synthesized silver nanoparticles (AgNPs) using aqueous leaf extract of Manilkara zapota to control Rhipicephalus (Boophilus) microplus. The synthesized AgNPs were characterized by UV-vis spectrum, scanning electron microscopy (SEM), Fourier transform infrared and X-ray diffraction. The UV-vis spectrum of the aqueous medium containing silver nanostructures showed a peak at 421 nm corresponding to the surface plasmon resonance band of AgNPs. SEM supports the biosynthesis and characterization of AgNPs with spherical and oval in shape and size of 70-140 nm. Acaricidal activity of aqueous leaf extract of M. zapota and synthesized AgNPs were carried out against R. (B.) microplus and the results showed the LC(50) values of 16.72 and 3.44 mg/L; r(2)=0.856 and 0.783), respectively.     

Susceptibility to two pyrethroids in Boophilus microplus (Acari: Ixodidae) populations of northwest Argentina. Preliminary results

Cattle are treated 6-12 times yearly to control Boophilus microplus ticks in the east zone of the Argentinean infested region, while 1-4 treatments are applied for tick control in the west zone. In the 1970s resistance to organo-phosphate acaricides was found in the east zone, but not in the west zone. However, a shift to synthetic pyrethroids (SP) was made through all infested regions. Currently, indications of resistance to SP in the east zone, but not in the west zone, are provoking to a switch to formamidine acaricides. During 1998 a total of 147 B. microplus engorged females were collected from 20 beef cattle ranches from the west zone of the Argentinean infested region. Individual progenies of these ticks were tested ('larval packet test') with cypermethrin and deltamethrin, and their LC 50 and LC 90 were compared to those estimated for the Milagro susceptible strain. No evidence of resistance to these SP was found. Due to sampling restraints the results are presented as preliminary. Nevertheless, a shift away from use of SP for control of B. microplus in the west zone appears to be unjustified and should be independent of the resistance circumstances observed in the east zone of the Argentinean tick infested region.     

Characterization and Purification of Glutathione S-Transferase from the Liver and Gill Tissues of Ağrı Balık Lake Trout Salmo trutta labrax and the Effects of Heavy Metal Ions on Its Activity

Glutathione S-transferase (GST) from the liver and gill tissues of A?r? Bal?k Lake Trout (also known as Black Sea Trout) Salmo trutta labrax was characterized and purified, and the toxic effects of some heavy metal ions on the enzyme's activity were analyzed. Liver GST was purified 930 times, resulting in 56% yield using glutathione–agarose affinity chromatography and a specific activity of 60.87 endotoxin units (EU)/mg protein. Using the same procedure, gill GST was purified 576 times, resulting in a 60% yield and specific activity of 46.8 EU/mg protein. The purity check of the purified enzymes was determined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Optimal pH, ionic strength, and stable pH were found for each tissue, and separate K_M and V_max values were determined for reduced glutathione and 2,4-dinitrochlorobenzene substrates. Heavy metal ions that have toxic effects on living organisms and are known to contribute to environmental pollution were selected, and their in vitro effects on enzyme activity were analyzed. The IC50 values and K_i constants of those metal ions showing an inhibitory effect on GST activity were determined.

Received November 24, 2014; accepted March 11, 2015     

The relationship between reduced glutathione level and glutathione S-transferase activity in sheep erythrocytes.

The relationship between reduced glutathione (GSH) level and glutathione S-transferase (GST) activity in erythrocytes was examined, using sheep erythrocytes, which have varying GSH concentrations, and dog erythrocytes with an inherited high concentration of GSH. There was a positive correlation (r = 0.529, p < 0.001) between the GSH level and GST activity in sheep erythrocytes. In dog erythrocytes, the GST activity in high-GSH cells was significantly (p < 0.001) higher than that in normal-GSH cells. These results indicate that the activity of GST in erythrocytes is directly correlated with the intracellular GSH level.     

Antioxidant defence system of boar cauda epididymidal spermatozoa and reproductive tract fluids

Antioxidants secreted by the reproductive tract protect spermatozoa against the toxic effects of reactive oxygen species (ROS) after ejaculation. This study aimed at characterizing the level of antioxidant protection in boar cauda epididymidal spermatozoa and fluids of the cauda epididymidis, vesicular and prostate glands. Also, this study investigated the effect of a 5-h period of dialysis on the antioxidant capacity of boar seminal plasma. Superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione transferase (GST) and phospholipid hydroperoxide glutathione peroxidase (PHGPx) activities were monitored in the cauda epididymidal spermatozoa or reproductive tract fluids. Also, the concentrations of total glutathione (GSH + GSSG), L-ergothioneine (ERT) and l-ascorbate and the total antioxidant status (TAS) of the fluids were measured. It was found that the cauda epididymidal spermatozoa exhibited high SOD activity and relatively low activity of PHGPx. The relative amounts of GPx, GR and GST activities in the cauda epididymidal spermatozoa were negligible, whereas CAT activity was undetectable. Greater SOD activity was found in the fluids of the cauda epididymidis and prostate gland. Furthermore, the prostate gland fluid appeared to be the main source of CAT activity in the seminal plasma, whereas the highest level of GPx activity was derived from the cauda epididymidal fluid. The reproductive tract fluids exhibited negligible amounts of GR and GST activities. It seemed that the significant amounts of GSH + GSSG, ERT and L-ascorbate in the reproductive tract fluids could have an ameliorative effect on the level of TAS in the seminal plasma. Dialysis had a marked effect on the total antioxidant capacity of the seminal plasma, which was manifested in greater activity of SOD and GPx. The findings of this study confirmed that the scavenging potential of the seminal plasma is dependent on the contributions of different antioxidants, originating in various fluids of boar reproductive tract.     

Enzymatic antioxidant defense in isolated rat hepatocytes exposed to cadmium

The aim of the study was the evaluation of cadmium effects on the activity of antioxidant enzymes in rat hepatocytes. The studies were conducted with isolated rat hepatocytes incubated for 1 or 2 hours in a modified (deprived of carbonates with phosphates) Williams' E medium (MWE) in the presence of cadmium chloride (25, 50 and 200 microM). Hepatocytes incubated in the MWE medium without cadmium chloride were used as a control. The application of the modified Williams' E medium allowed for the appearance of cadmium compounds in a soluble form that is indispensable for suitable estimation of its toxic action. There were evaluated markers of the oxidative stress such as: concentration of thiobarbiturate reactive substances (TBARS)--proportional to the level of lipid peroxidation, concentration of reduced glutathione (GSH), and the activity of antioxidant enzymes, including superoxide dismutase (SOD1 and SOD2), catalase (CAT), total glutathione peroxidase (GSHPx), selenium--dependent glutathione peroxidase (SeGSHPx), glutathione transferase (GST) and glutathione reductase (GSHR). Alterations of antioxidant enzymes activity, the level of TBARS and GSH in isolated rat hepatocytes caused by cadmium in vitro, were shown to depend on the concentration and time of exposure of cells to this metal. The increased level of TBARS and GSH was observed as well as changes in the activity of antioxidant enzymes. The activity of SOD isoenzymes and CAT was increased, whereas GSHPx and GST were decreased. These results indicate that cadmium induces oxidative stress followed by alterations in the cellular antioxidant enzyme system in isolated rat hepatocytes.     

A new in vitro test to evaluate the resistance level against acaricides of the cattle tick, Rhipicephalus (Boophilus) microplus

In this article we present a new bioassay to assess the resistance status of ticks to acaricides. The Larval Tarsal Test (LTT) is a sensitive, highly time-effective in vitro test. It allows the investigation of a large number of compounds and doses on the cattle tick Rhipicephalus (Boophilus) microplus in a short period of time. The ability of the LTT to assess the lethal concentration at 50% mortality (LC(50)) and resistance ratios (RRs) of a susceptible and a resistant R. microplus strain was compared with the FAO-recommended Larval Packet Test (LPT). Representative compounds of the carbamate, organophosphate (OP), synthetic pyrethroid (SP), formamidine (FOR), macrocyclic lactone and pyrazole classes were used for this comparison. The resistance status against OP, SP and FOR of the resistant R. microplus strain was confirmed in vivo. The LTT resulted in resistance ratios comparable to those obtained with the LPT. However, the lethal concentrations were up to 150-fold lower in the LTT than in the LPT. The advantage of the LTT is to simplify the methodology by avoiding the handling of larvae and using multi-well plates. The LTT is therefore a suitable test for the assessment of the level of resistance of R. microplus and is very promising to evaluate the resistance profile of field strains. Additionally, the LTT is also suitable to test other ixodid species.     

Molecular cloning, expression and characterization of a functional GSTmu class from the cattle tick Boophilus annulatus

A full-length cDNA of a glutathione S-transferase (GST) was cloned from a cDNA library of the local Egyptian cattle tick Boophilus annulatus. The 672 bp cloned fragment was sequenced and showed an open reading frame encoding a protein of 223 amino acids. Comparison of the deduced amino acid sequence with GSTs from other species revealed that the sequence is closely related to the mammalian mu-class GST. The cloned gene was expressed in E. coli under T7 promotor of pET-30b vector, and purified under native conditions. The purified enzyme appeared as a single band on 12% SDS-PAGE and has a molecular weight of 30.8 kDa including the histidine tag of the vector. The purified enzyme was assayed upon the chromogenic substrate 1-chloro-2,4-dinitrobenzene (CDNB) and the recombinant enzyme showed high level of activity even in the presence of the beta-galactosidase region on its 5' end and showed maximum activity at pH 7.5. The Km values for CDNB and GSH were 0.57 and 0.79 mM, respectively. The over expressed rBaGST showed high activity toward CDNB (121 units/mg protein) and less toward DCNB (29.3 units/mg protein). rBaGST exhibited peroxidatic activity on cumene hydroperoxide sharing this property with GSTs belonging to the GST alpha class. I50 values for cibacron blue and bromosulfophthalein were 0.22 and 8.45 microM, respectively, sharing this property with the mammalian GSTmu class. Immunoblotting revealed the presence of the GST molecule in B. annulatus protein extracts; whole tick, larvae, gut, salivary gland and ovary. Homologues to the GSTmu were also detected in other tick species as Hyalomma dromedarii and Rhipicephalus sp. while in Ornithodoros moubata, GSTmu homologue could not be detected.     

滞育发动前家蚕滞育性与非滞育性卵的谷胱甘肽氧化还原循环状态分析

为了调查滞育和非滞育性家蚕卵的谷胱甘肽氧化还原循环在滞育发动前是否存在差异,利用分光光度法检测滞育发动前(25℃中蚕卵产下后0～24 h)滞育与非滞育蚕卵的谷胱甘肽含量和相关代谢酶活性。滞育发动前,滞育性家蚕卵中的还原型谷胱甘肽(GSH)含量、氧化型谷胱甘肽(GSSG)含量、总谷胱甘肽(GSH+2GSSG)含量和GSH/GSSG比值变化不显著,谷胱甘肽S-转移酶(GST)活性变化不显著,但硫氧还蛋白过氧化物酶(TPX)活性下降23%,谷胱甘肽还原酶(GR)活性升高57%;非滞育性家蚕卵中的GSH含量及TPX和GST活性变化不显著,但GSSG含量升高61%,GSH+2GSSG含量升高41%,GSH/GSSG比值下降33%,GR活性下降16%。与非滞育性家蚕卵相比,滞育发动前滞育性家蚕卵中的GSH含量、GSSG含量和GSH+2GSSG含量较低,GSH/GSSG比值较高,TPX活性较低,GST活性无显著差异,GR活性较高。两种蚕卵中都未能检测到谷胱甘肽过氧化物酶(GPX)和硫氧还蛋白还原酶(TrxR)活性。结果表明,滞育发动前滞育性家蚕卵中较低的TPX活性和较高的GR活性共同导致较高的GSH/GSSG比值,使其谷胱甘肽氧化还原循环处于相对还原态。     

In vitro and in vivo evaluation of deltamethrin and amitraz mixtures for the control of Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) in New Caledonia

Acaricide resistance is a major problem that hinders the control of the tropical cattle tick, Rhipicephalus (Boophilus) microplus (Canestrini), in many parts of the world where cattle production continues to suffer severe economic losses to tick infestation. Deltamethrin and amitraz have been used alone to control R. microplus in New Caledonia for the past decade, and tick populations have developed resistance to both acaricides. A study was conducted to evaluate the effectiveness of deltamethrin and amitraz mixtures, through in vitro laboratory bioassays and in vivo on-animal efficacy trials, for the control of resistant R. microplus on cattle at two dairy farms in New Caledonia. Results of laboratory bioassays using modified larval packet tests (LPT) revealed up to 16.59-fold resistance to deltamethrin, and up to 5.86-fold resistance to amitraz. Significant synergism was observed when amitraz was used as a synergist in deltamethrin bioassays. Amitraz significantly increased deltamethrin toxicity to tick larvae, while deltamethrin was much less effective on amitraz toxicity. Synergism of amitraz by deltamethrin only occurred when the deltamethrin concentration was relatively high. Results of on animal efficacy trials of deltamethrin and amitraz alone and mixtures of both at different concentrations revealed a similar pattern of synergism. Adding amitraz to a deltamethrin formulation led to dramatic increases of percent reduction of both immature and adult ticks. In contrast, adding deltamethrin to an amitraz formulation did not increase control efficacy. Results from this study may lead to the adoption of an acaricide mixture strategy for the control of pyrethroid-resistant R. microplus in New Caledonia and elsewhere.     

Tests to determine LC50 and discriminating doses for macrocyclic lactones against the cattle tick, Boophilus microplus

Laboratory tests were carried out on larvae and adults of the cattle tick Boophilus microplus to determine the toxicity of macrocyclic lactone acaricides (MLs). Technical and commercial MLs were used in larval packet test (LPT), larval immersion test (LIT) and adult immersion test (AIT). In LIT and AIT the toxicity of MLs was much higher than for LPT. In the AIT, diluting the injectable formulation of MLs in water was as effective as dilution in ethanol+Triton X-100. LC50, LC99.9 and 95% confidence limits were determined so that a discriminating dose (DD) could be set for larval and adult tests in order to diagnose potential resistance to MLs in field samples of the tick. These DDs are for Australian strains of B. microplus and may not be suitable for other strains until further work is carried out.The value of these diagnostic tests can only be verified if or when resistance to MLs emerges in ticks.     

Susceptibility of Brazilian samples ofBoophilus microplus to organophosphorus acaricides

This paper describes the susceptibility of various samples of Boophilus microplus from the southern region of Minas Gerais State in Brazil to the 4 commonest organophosphorus acaricides employed in the area. All samples showed some degree of resistance to each of the compounds used. The resistance factor varied from 1.42 to 132.90, being less in the case of dicrotophos fenthion than with chlorpyrifos and coumaphos.     

家蚕氟化物中毒后血淋巴中脂质过氧化物和抗氧化物含量及抗氧化酶活性的变化

为了探究氟化物对家蚕的毒理作用,通过给家蚕5龄期幼虫添食NaF,检测氟化物中毒家蚕幼虫血淋巴中脂质过氧化物质丙二醛(MDA)、抗氧化物质谷胱甘肽(GSH)的含量变化以及谷胱甘肽过氧化物酶(GSH-Px)、谷胱甘肽硫转移酶(GST)等抗氧化物酶的活性变化。正常家蚕幼虫和氟化物中毒家蚕幼虫血淋巴中的MDA含量在5龄期均逐渐下降,但氟化物中毒家蚕的MDA平均浓度比正常家蚕升高了83.77%;GSH含量变化趋势为5龄第1天和第5天较高,第3天较低,但氟化物中毒家蚕5龄期的GSH浓度平均比正常家蚕下降了47.81%;氟化物中毒家蚕5龄期的GSH-Px和GST的平均活性分别比正常家蚕升高63.49%、39.22%,5龄第3天达到最高峰,5龄第1天和第5天较弱。初步分析认为家蚕幼虫血淋巴中MDA和GSH的含量变化以及GSH-Px和GST的活性变化,与家蚕氟化物中毒后体内氧自由基含量上升和脂质过氧化作用增强密切相关,可作为诊断家蚕氟化物中毒的生化指标。     

柞蚕饰腹寄蝇不同地理种群的杀虫剂靶标酶和解毒酶活性分析

昆虫通过降低靶标酶敏感性及增强对杀虫剂的代谢能力产生抗药性。蝇毒磷是防治柞蚕饰腹寄蝇(Blepharipa tibia-lis Chao)的常用有机磷杀虫剂,为探讨柞蚕饰腹寄蝇不同地理种群对蝇毒磷的抗药性分化程度,分析来自辽宁省瓦房店市、凤城市、庄河市、开原市和西丰县的5个柞蚕饰腹寄蝇地理种群体内的乙酰胆碱酯酶(AChE)、谷胱甘肽S-转移酶(GST)、细胞色素P450(CYP450)和羧酸酯酶(CarE)4种酶的活性及蝇毒磷对这些酶活性的体外抑制率。结果表明:来自瓦房店市和凤城市的柞蚕饰腹寄蝇地理种群的AChE比活力及蝇毒磷对酶活性的体外抑制率均显著高于其它3个地理种群;瓦房店市地理种群的GST比活力显著高于其它4个地理种群,蝇毒磷对酶活性的体外抑制率显著高于除庄河市地理种群外的其它3个地理种群;庄河市地理种群的CYP450比活力显著高于来自瓦房店市、凤城市和西丰县的3个地理种群,但与开原市地理种群相比差异不显著,蝇毒磷对瓦房店市地理种群的CYP450活性的体外抑制率显著高于其它4个地理种群;瓦房店市与凤城市地理种群的CarE比活力均显著高于来自庄河市、开原市和西丰县的3个地理种群;蝇毒磷对瓦房店市和凤城市地理种群的CarE活性的体外抑制率显著高于庄河市地理种群,对其它地理种群间的CarE活性的体外抑制率差异不显著。根据以上检测结果,推测来自庄河市的柞蚕饰腹寄蝇地理种群对蝇毒磷的抗性相对较强,而来自瓦房店市的柞蚕饰腹寄蝇地理种群对蝇毒磷的抗性相对较弱。     

Compatibility of the fungus Metarhizium anisopliae and deltamethrin to control a resistant strain of Boophilus microplus tick

The tick Boophilus microplus causes economic impact to cattle producers and has a great capacity to develop resistance to chemical acaricides. It is very important the development of new techniques to complement the control of this parasitosis. Biological control is a promising option to maintain acceptable levels of tick populations in cattle. Therefore, the present study evaluated the association of deltamethrin and the entomopathogenic fungus Metarhizium anisopliae against B. microplus larvae resistant to pyrethroid. The synthetic pyrethroid, deltamethrin, was used at concentrations of 0.39, 0.78, 1.56, 3.12 and 6.12 ppm, M. anisopliae was used at concentrations of 10(5), 10(6), 10(7) and 10(8) conidia ml(-1). Their associations were also evaluated in in vitro tests. The assays showed that the tick strain is resistant to deltamethrin, but high mortality rates were observed when deltamethrin was associated with the entomopathogen. The larvae's mortality rates ranged from 7 to 36.5% for the different concentrations of deltamethrin, however, for the different concentrations of M. anisopliae, the mortality rates ranged from 10 to 96.9%. Mortality rates were proportional to the concentrations used for both deltamethrin and the fungus. Predominantly, the association of pyrethroid and fungus resulted in higher larvae mortalility rates than those obtained with the respective non-associated concentrations. Thus, the present study indicates that this association can be used as a tool for integrated control of the tick B. microplus.     

家蚕谷胱甘肽-S-转移酶E4基因的组织表达和序列特征及重组蛋白的酶活性分析

昆虫体内的谷胱甘肽-S-转移酶(GSTs)主要行使对异生或内源有毒物质进行代谢解毒的功能。选择家蚕谷胱甘肽-S-转移酶E4的编码基因Bmgste4为靶标,研究其组织表达谱、序列结构特征及原核表达重组蛋白的酶活性。RT-PCR检测不同组织表达特征的结果显示,Bmgste4基因在家蚕5龄第3天幼虫的头部、触角、下颚须、表皮、脂肪体、丝腺以及雄性个体的马氏管和雌性个体的卵巢中均有表达,而在血细胞、中肠以及雄性个体的精巢和雌性个体的马氏管中不表达。多序列比对结果显示BmGSTE4具有完整和保守的GST二级结构特征,N端氨基酸残基保守,特别是谷胱甘肽结合位点。构建插入Bmgste4基因开放阅读框片段的重组表达质粒p28-Bmgste4,并转化E.coli Rosetta(DE3)感受态细胞进行原核表达后,利用分光光度法测定重组蛋白对通用底物1-氯-2,4-二硝基苯的酶活参数:Km=0.58 mmol/L,Vmax=24.55μmol/(mg.min)。这些结果为进一步研究家蚕体内解毒和抗药性机制以及开发鳞翅目害虫新型生物防治药剂提供了基础信息。     

二化性家蚕滞育过程中谷胱甘肽S-转移酶活性的变化

二化性家蚕卵以25℃和15℃催青,可分别诱导成虫产下子代滞育和非滞育卵,而即时浸酸和5℃左右的低温处理可以分别阻止和解除胚胎滞育。利用1-氯-2,4-二硝基苯(CDNB)显色法测定了经上述处理后的胚胎滞育过程中谷胱甘肽S-转移酶(glutathione S-transferase,GST)的活性,结果表明:胚胎发育后期的蚕卵经25℃催青,卵中的GST活性显著高于15℃催青蚕卵;蚕卵即时浸酸未显著改变胚胎滞育发动阶段蚕卵中的GST活性;5℃低温处理显著提高了滞育卵中的GST活性,但是未能显著改变即时浸酸卵的GST活性。上述结果表明,蚕卵中的GST活性变化与二化性家蚕胚胎滞育诱导和解除密切相关。     

Serologically defined Rhipicephalus (Boophilus) microplus larval antigens in BmLF3, a partially pure Sephacryl S-300 fraction of crude larval proteins

This report is designed to provide additional information regarding larval soluble proteins toward the planned development of a comprehensive database of Rhipicephalus (Boophilus) microplus proteins that elicit a humoral immune response in cattle as a result of natural ectoparasite infestation. Larval proteins of R. microplus are complex and the protein profile is not dominated by any major proteins. This report focuses upon an S-300 Sephacryl (molecular sieve) column fraction, fraction 3 (BmLF3). With the use of SDS-PAGE (without-2ME) and Western blotting with a composite pool of pre- and post-R. microplus larval infestation antiserum BmLF3 was found to contain 7 apparent common ixodid major antigens (207.3, 171.9, 98.0, 86.5, 65.7, 58.9, and 38.0 kDa), those potentially shared with other ixodid species, and 2 apparent R. microplus specific antigens evidenced by low-level antibody binding in crude BmLF3 (149.4 kDa) and HPLC peak 8 of BmLF3 (116.0 kDa). In addition, BmLF3 contains potent inhibitors of trypsin activity. However, these inhibitors of trypsin did not appear to elicit host antibodies as a result of natural ectoparasite exposure, as defined by Western blotting of reduced and denatured trypsin binding proteins purified by affinity chromatography.