Application of Vascular Puncture for Evaluation of Curtovirus Resistance in Chile Pepper and Tomato

Curtoviruses cause severe damage to tomatoes and peppers. Functional field resistance to curtoviruses in these plants is desirable but difficult to produce and difficult to screen for because it is time‐consuming and resistance could be achieved by developing resistance either to the virus or to insect feeding. To improve and speed curtovirus resistance testing in tomato (Solanum lycopersicum) and pepper (Capsicum annuum) plants, two puncture methods were developed and compared to leafhopper inoculation and feeding preference assays. The two puncture methods were adapted to introduce a modified Agrobacterium tumefaciens plasmid carrying a recombinant curtovirus into the meristem tissue of tomato plants and into newly germinated chile pepper seedlings. The puncture techniques were used to screen for resistance to curtoviruses in chile pepper and tomato breeding lines and varieties. Similarly, the peppers and tomatoes were assayed for curtovirus resistance using leafhopper inoculation and feeding preference, which was assessed by stylet sheath staining. Virus infection by puncture and leafhopper feeding was monitored using PCR and ELISA. ELISA was performed using an antibody to bacterially expressed coat protein. While pepper cvs Tabasco, NuMex Las Cruces cayenne and New Mexico 6‐4 were infected using both puncture and leafhopper inoculation methods, New Mexico 6‐4 had higher infection rates than the other two cultivars. Stylet sheath staining results suggest that leafhoppers prefer to feed on New Mexico 6‐4 rather than Tabasco and NuMex Las Cruces cayenne. Eight tomato cultivars were infected using meristem removal injection inoculation. Three tomatoes cultivars (CVF‐11, Saladmaster and Supersteak) were infected using leafhopper inoculation, although stylet sheath staining results suggested that the first two cultivars were not preferred by the insect vector. Our results suggest that puncture methods and leafhopper inoculation are successful in resistance screening, and both methods should be used as part of screening, because they assess different types of resistance.     

PBAN receptor: Employment of anti-receptor antibodies for its characterization and for development of a microplate binding assay

This study describes generation of an anti-PBAN receptor (PBAN-R) antiserum and its employment for the characterization of the PK/PBAN-R(s). The antiserum recognized, in a specific and dose-dependent manner, the presence of PBAN-R in pheromone gland membrane preparations of three female moths: Heliothis peltigera, Helicoverpa armigera and Spodoptera littoralis. It also reacted specifically with the S. littoralis larval receptor in vivo, most likely by competing with the ligand on the binding site and consequently inhibiting cuticular melanization. Despite its ability to react with the receptor of H. peltigera in dot blot experiments, the antiserum did not react with the receptor in vivo and failed to inhibit sex pheromone biosynthesis. The antiserum was also used to develop two microplate binding assays. The Ab described in this study is the first raised against an insect neuropeptide (Np) receptor to be used in vivo, and its employment for characterization of the PK/PBAN-R(s) may thus provide important information on the mode of action of this Np family. The present study adds important information on the difference between the receptors in the two moth species, hints at the possible existence of receptor subtypes, and provides a platform for the development of a high-throughput assay (HTA) for screening of PK/PBAN agonists and antagonists.     

间斑寇蛛 L.tredecimguttatus粗毒抗血清制备及其活性分析

间斑寇蛛Latrodectus tredecimguttatus是世界上毒性最强的蜘蛛之一,其毒液中含有许多大分子物质,可用于制备抗血清。将粗毒过滤除菌后作为抗原,加弗氏佐剂背部皮下免疫豚鼠。加强免疫3次后经检测抗血清效价达到1：8,再次加强免疫后断颈处死豚鼠放血,分离血清,经饱和硫酸铵分级沉淀及阴离子交换柱层析纯化后,采用westernblot法检测抗体所在峰。离体大鼠输精管电刺激收缩反应实验及活体动物给药实验均表明抗体对毒素具有明显的中和作用。特异性间斑寇蛛毒素抗血清为治疗间斑寇蛛咬伤奠定了基础。     

Identification of enteropathogenic and enterohaemorrhagic Escherichia coli strains by immunoserological detection of intimin

Aims: To evaluate the sensitivity and specificity of polyclonal and monoclonal antibodies (Mabs) against intimin in the detection of enteropathogenic and enterohaemorrhagic Escherichia coli isolates using immunoblotting. Methods and Results: Polyclonal and Mabs against the intimin‐conserved region were raised, and their reactivities were compared in enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli (EHEC) isolates using immunoblotting analysis. In comparison with rat antiserum, rabbit anti‐intimin IgG‐enriched fraction had a stronger recognition pattern to a wide spectrum of intimin types in different EPEC and EHEC serotypes. On the other hand, murine monoclonal IgG2b specific to intimin, with dissociation constant of 1·3 × 10^?8 mol l^?1, failed in the detection of some of these isolates. Conclusion: All employed antibodies showed 100% specificity, not reacting with any of the eae‐negative isolates. The sensitivity range was according to the employed antisera, and 97% for rabbit anti‐intimin IgG‐enriched fraction, followed by 92% and 78% sensitivity with rat antisera and Mab. Significance and Impact of the Study: The rabbit anti‐intimin IgG‐enriched fraction in immunoblotting analysis is a useful tool for EPEC and EHEC diagnoses.     

The biological role of pituitary adenylate cyclase‐activating polypeptide (PACAP) in growth and feeding behavior in juvenile fish

To date, many technologies have been developed to increase efficiency in aquaculture, but very few successful biotechnology molecules have arrived on the market. In this context, marine biotechnology has an opportunity to develop products to improve the output of fish in aquaculture. Published in vivo studies on the action of the pituitary adenylate cyclase‐activating polypeptide (PACAP) in fish are scarce. Recently, our group, for the first time, demonstrated the biological role of this neuropeptide administrated by immersion baths in the growth and development of larval fish. In this work, we have evaluated the effects of recombinant Clarias gariepinus PACAP administration by intraperitoneal injection on growth performance and feeding behavior in juvenile fish. Our results showed the physiological role of this peptide for growth control in fish, including the juvenile stage, and confirm that its biological functions are well conserved in fish, since C. gariepinus PACAP stimulated growth in juvenile tilapia Oreochromis niloticus. In addition, we have observed that the growth‐promoting effect of PACAP in juvenile tilapia was correlated with higher GH concentration in serum. With regard to the neuroendocrine regulation of growth control by PACAP, it was demonstrated that PACAP stimulates food intake in juvenile tilapia. In general, PACAP appears to act in the regulation of the growth control in juvenile fish. These findings propose that PACAP is a prominent target with the potential to stimulate fish growth in aquaculture. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Concerted simulations reveal how peroxidase compound III formation results in cellular oscillations

A major problem in mathematical modeling of the dynamics of complex biological systems is the frequent lack of knowledge of kinetic parameters. Here, we apply Brownian dynamics simulations, based on protein three-dimensional structures, to estimate a previously undetermined kinetic parameter, which is then used in biochemical network simulations. The peroxidase-oxidase reaction involves many elementary steps and displays oscillatory dynamics important for immune response. Brownian dynamics simulations were performed for three different peroxidases to estimate the rate constant for one of the elementary steps crucial for oscillations in the peroxidase-oxidase reaction, the association of superoxide with peroxidase. Computed second-order rate constants agree well with available experimental data and permit prediction of rate constants at physiological conditions. The simulations show that electrostatic interactions depress the rate of superoxide association with myeloperoxidase, bringing it into the range necessary for oscillatory behavior in activated neutrophils. Such negative electrostatic steering of enzyme-substrate association presents a novel control mechanism and lies in sharp contrast to the electrostatically-steered fast association of superoxide and Cu/Zn superoxide dismutase, which is also simulated here. The results demonstrate the potential of an integrated and concerted application of structure-based simulations and biochemical network simulations in cellular systems biology.     

Screening of in Vitro Inhibition of Lactoperoxidase Enzyme by Methyl Benzoate Derivatives with Molecular Docking Studies

Lactoperoxidase enzyme (LPO) is secreted from salivary, mammary, and other mucosal glands including the bronchi, lungs, and nose, which had functions as a natural and the first line of defense towards viruses and bacteria. In this study, methyl benzoates were examined in LPO enzyme activity. Methyl benzoates are used as precursors in the synthesis of aminobenzohydrazides used as LPO inhibitors. For this purpose, LPO was purified in a single step using sepharose-4B-l-tyrosine-sulfanilamide affinity gel chromatography with a yield of 9.91 % from cow milk. Also, some inhibition parameters including the half maximal inhibitory concentration (IC₅₀) value and an inhibition constant (K_i) values of methyl benzoates were determined. These compounds inhibited LPO with K_i values ranging from 0.033±0.004 to 1540.011±460.020 μM. Compound 1 a (methyl 2-amino-3-bromobenzoate) showed the best inhibition (K_i=0.033±0.004 μM). The most potent inhibitor ( 1 a ) showed with a docking score of −3.36 kcal/mol and an MM-GBSA value of −25.05 kcal/mol, of these methyl benzoate derivatives ( 1 a – 16 a ) series are established H-bond within the binding cavity with residues Asp108 (distance of 1.79 Å), Ala114 (distance of 2.64 Å), and His351 (distance of 2.12 Å).     

促甲状腺激素抗血清的研制

用较少的抗原量免疫绵羊,采用活体采血法,定期加强免疫,定期补铁,定期采血,获得抗人促甲状腺激素(TSH)抗血清.采用放免分析方法鉴定抗血清,结果为:抗血清滴度为28-205万,与促卵泡激素、促绒毛膜生长激素、促黄体生成素交叉都小于0.02‰,亲和常数K大于10¹⁰L/mol.