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1.
Wheat germ agglutinin (WGA) is a plant lectin, which specifically recognizes the sugars NeuNAc and GlcNAc. Mutated WGA with enhanced binding specificity can be used as biomarkers for cancer. In silico mutations are performed at the active site of WGA to enhance the binding specificity towards sialylglycans, and molecular dynamics simulations of 20 ns are carried out for wild type and mutated WGAs (WGA1, WGA2, and WGA3) in complex with sialylgalactose to examine the change in binding specificity. MD simulations reveal the change in binding specificity of wild type and mutated WGAs towards sialylgalactose and bound conformational flexibility of sialylgalactose. The mutated polar amino acid residues Asn114 (S114N), Lys118 (G118K), and Arg118 (G118R) make direct and water mediated hydrogen bonds and hydrophobic interactions with sialylgalactose. An analysis of possible hydrogen bonds, hydrophobic interactions, total pair wise interaction energy between active site residues and sialylgalactose and MM‐PBSA free energy calculation reveals the plausible binding modes and the role of water in stabilizing different binding modes. An interesting observation is that the binding specificity of mutated WGAs (cyborg lectin) towards sialylgalactose is found to be higher in double point mutation (WGA3). One of the substituted residues Arg118 plays a crucial role in sugar binding. Based on the interactions and energy calculations, it is concluded that the order of binding specificity of WGAs towards sialylgalactose is WGA3 > WGA1 > WGA2 > WGA. On comparing with the wild type, double point mutated WGA (WGA3) exhibits increased specificity towards sialylgalactose, and thus, it can be effectively used in targeted drug delivery and as biological cell marker in cancer therapeutics. Copyright © 2014 John Wiley & Sons, Ltd.  相似文献   
2.
Bacillus sp. YX-1 glucose dehydrogenase (BsGDH) with good solvent resistance catalyzes the oxidation of β-d-glucose to d-glucono-1,5-lactone. Xylose is a recyclable resource from hemicellulase hydrolysis. In this work, to improve the preference of BsGDH for xylose, we designed seven mutants inside or adjacent to the substrate binding pocket using site-directed mutagenesis. Among all mutants, Ala258Phe mutant displayed the highest activity of 7.59 U mg−1 and nearly 8-folds higher kcat/Km value towards xylose than wild-type BsGDH. The kinetic constants indicated that the A258F mutation effectively altered the transition state. By analysis of modeled protein structure, Ala258Phe created a space to facilitate the reactivity towards xylose. A258F mutant retained good solvent resistance in glycol, ethyl caprylate, octane, decane, cyclohexane, nonane, etc. as with BsGDH. This work provides a protein engineering approach to modify the substrate stereo-preference of alcohol dehydrogenase and a promising enzyme for cofactor regeneration in chiral catalysis.  相似文献   
3.
We determined the host range of the parasitoid Trichomalus perfectus (Walker), a candidate for classical biological control of cabbage seedpod weevil, Ceutorhynchus obstrictus (Marsham), an important pest of canola in Canada. Studies were conducted in Europe and in North America. In laboratory experiments, the levels of parasitism (acceptance) of Ceutorhynchus turbatus Schultze, C. cardariae Korotyaev, C. omissus Fall and C. querceti (Gyllenhal) by T. perfectus were not significantly different than of the target host C. obstrictus. Although C. typhae (Herbst), C. pallidactylus (Marsham), C. americanus Buchanan, C. neglectus Blatchely and Ceutorhynchus sp. nr. nodipennis were parasitised by T. perfectus, the levels of parasitism were significantly lower on these species than on C. obstrictus. Ceutorhynchus peyerimhoffi Hustache, C. erysimi (Fabricius), C. alliariae H. Brisout, C. roberti Gyllenhal, Mogulones borraginis (Fabricius), Mononychus vulpeculus (Fabricius) and the leaf-mining fly Scaptomyza flava (Fallén) were not attacked. Ecological host range surveys in Europe corroborated the prediction that T. perfectus would attack C. cardariae at similar rates to C. obstrictus. In North America, the recent discovery of T. perfectus in a C. omissus population suggests that laboratory findings predicting that C. omissus is a preferred host may be the case in the field. We found that T. perfectus attacks larvae of some Ceutorhynchus spp. feeding on Brassicaceae and does not attack species outside of that host range. Thus, the parasitoid can be defined as narrowly oligophagous. These results demonstrate the value of ecological host range studies in the area of origin to validate hypotheses generated through laboratory host range experiments.  相似文献   
4.
Selitrichodes neseri (Hymenoptera: Eulophidae) is a parasitoid of the invasive gall-forming wasp Leptocybe invasa (Hymenoptera: Eulophidae), which has caused serious damage to Eucalyptus plantations in many parts of the world. S. neseri is a recently discovered parasitoid considered to be a potentially important biological control agent of L. invasa. The aim of this study was to provide the first basic data on the biology of S. neseri, which is essential for its application in biological control. S. neseri was shown to be a biparental ectoparasitoid. Observation from dissected galls indicated that the parasitoid developed on late larvae, pupae and callow adults, although development did occur in a range of gall ages. Observed nominal parasitism in captivity ranged from 9.7% to 71.8%. Adult S. neseri specimens, fed with honey-water and galled Eucalyptus leaves, survived an average of 26 days at 26 °C. The average developmental time from oviposition to emergence was 19.3 days ± 0.2 days. There was no pre-oviposition period. A single female produced a maximum of thirty-nine offspring, with a maximum of ten per day. Dissection of the ovaries showed that twelve ovarioles were present. The sex ratio of S. neseri observed in this study was 1:3.43 males:females. Galls of native insects most closely related to L. invasa and to galls of similar morphology to L. invasa-induced galls, were not suitable for S. neseri oviposition. S. neseri showed considerable potential as a biological control agent of L. invasa due to its relatively short developmental time, long adult life span when supplemented with carbohydrates, ability to utilize a range of gall ages and the fact that it has a high level of host specificity.  相似文献   
5.
Summary In previous studies we evaluated the incidence and specificity of autologous antibody reactivity against squamous cell carcinoma of the head and neck (SCCHN). We were able to demonstrate that autologous antibody reactivity is present in native sera but was usually of too low a titer to allow further analysis. Dissociation of immune complexes by acidification and ultrafiltration of serum augmented autologous antibody reactivity in nine out of nine autologous systems tested. Native antibody and antibody derived from immune complexes produced by the host and reactive with autologous tumor cells may be directed against physiologically relevant antigens. Therefore, correlations of antibody titers with clinical course may provide insight into the nature of the host response to cancer. In the present analysis, serological studies of six patients with SCCHN were performed with serum samples obtained over many months. Results of serial serological assays were correlated to tumor progression and clinical course. Fluctuations in autologous antibody reactivity were noted over time. In four cases, rises in autologous antibody titers preceded the clinical diagnosis of recurrence by several months. Drops in autologous antibody reactivity were noted in two cases following surgery or radiation therapy. In two cases of long-term survivors, no correlation between antibody reactivity and clinical course was noted. Specificity analysis of the six autologous systems demonstrated reactivity against autologous and allogeneic SCCHN as well as melanoma cell lines. These sera did not react with glioma, neuroblastoma, renal cell, breast, bladder and colon carcinoma cell lines nor with fetal calf serum, pooled lymphocytes, red blood cells and platelets. Autologous serial serological studies may provide a means by which to evaluate the host/tumor relationship in patients with SCCHN.  相似文献   
6.
Molecular recognition is central to biology and ranges from highly selective to broadly promiscuous. The ability to modulate specificity at will is particularly important for drug development, and discovery of mechanisms contributing to binding specificity is crucial for our basic understanding of biology and for applications in health care. In this study, we used computational molecular design to create a large dataset of diverse small molecules with a range of binding specificities. We then performed structural, energetic, and statistical analysis on the dataset to study molecular mechanisms of achieving specificity goals. The work was done in the context of HIV‐1 protease inhibition and the molecular designs targeted a panel of wild‐type and drug‐resistant mutant HIV‐1 protease structures. The analysis focused on mechanisms for promiscuous binding to bind robustly even to resistance mutants. Broadly binding inhibitors tended to be smaller in size, more flexible in chemical structure, and more hydrophobic in nature compared to highly selective ones. Furthermore, structural and energetic analyses illustrated mechanisms by which flexible inhibitors achieved binding; we found ligand conformational adaptation near mutation sites and structural plasticity in targets through torsional flips of asymmetric functional groups to form alternative, compensatory packing interactions or hydrogen bonds. As no inhibitor bound to all variants, we designed small cocktails of inhibitors to do so and discovered that they often jointly covered the target set through mechanistic complementarity. Furthermore, using structural plasticity observed in experiments, and potentially in simulations, is suggested to be a viable means of designing adaptive inhibitors that are promiscuous binders. Proteins 2015; 83:351–372. © 2014 Wiley Periodicals, Inc.  相似文献   
7.
The activity of 1-acylglycerophosphocholine (1-acyl-GPC) O-acyltransferase (EC 2.3.1.23) varied during maturation of safflower (Carthamus tinctorius L.) seeds, and activity per seed was highest in the middle period of seed development when triacylglycerol (TAG) is most rapidly synthesized. The specific activity of acyl transfer in a 20000·g particulate preparation exceeded 500nmol·min-1·(mg protein)-1 and was higher than those of any other enzymes involved in TAG synthesis (K. Ichihara et al., 1993, Plant Cell Physiol. 34, 557–566). This suggested the presence of a large flux of acyl-CoA to phosphatidylcholine in the cell. The reaction was specific to C16 and C18 acyl-CoAs with a double bond at position 9. Lauroyl- and erucoyl-CoA were completely ineffective, while ricinoleoyl- and elaidoyl-CoA were utilized efficiently. The relative order of specificity for native acyl-CoA species was linoleoyl > oleoyl stearoyl = palmitoyl. When acyl-CoA mixtures were presented, preference for the unsaturated species rather than the saturated species was even more apparent. The enzyme preferentially utilized 1-C16-acyl- and 1-C18-acyl-GPC molecular species, and 1-palmitoyl-, 1-stearoyl-, 1-oleoyl-and 1-linoleoyl-GPC equally served as acyl acceptor. No activity was detected with 1-octanoyl-GPC, and 1-erucoyl-GPC produced little effect. The effectiveness of 1-alkyl-GPC was comparable to that of 1-acyl-GPC. It was thus concluded that the enzyme recognizes the chain lengths of the acyl donor and acceptor, and the double bond at position 9 of the acyl donor.Abbreviations DAG diacylglycerol - DTNB 5,5-dithiobis(2-nitrobenzoic acid) - GP sn-glycerol 3-phosphate - GPC sn-glycero-3-phosphocholine - GPE sn-glycero-3-phosphoethanolamine - GPI sn-glycero-3-phosphoinositol - PC phosphatidylcholine - TAG triacylglycerol  相似文献   
8.
Abiotic environmental heterogeneity can promote the evolution of diverse resource specialists, which in turn may increase the degree of host–parasite specialization. We coevolved Pseudomonas fluorescens and lytic phage ?2 in spatially structured populations, each consisting of two interconnected subpopulations evolving in the same or different nutrient media (homogeneous and heterogeneous environments, respectively). Counter to the normal expectation, host–parasite specialization was significantly lower in heterogeneous compared with homogeneous environments. This result could not be explained by dispersal homogenizing populations, as this would have resulted in the heterogeneous treatments having levels of specialization equal to or greater than that of the homogeneous environments. We argue that selection for costly generalists is greatest when the coevolving species are exposed to diverse environmental conditions and that this can provide an explanation for our results. A simple coevolutionary model of this process suggests that this can be a general mechanism by which environmental heterogeneity can reduce rather than increase host–parasite specialization.  相似文献   
9.
The trap crop strategy is based on host plant discrimination by pests and their parasitoids, which may respond differently to various host plant cues, thus affecting their respective population distributions. We conducted a three-year study to compare the responses of the most damaging pest of oilseed rape (Brassica napus L.), the pollen beetle (Meligethes aeneus Fab.), and its hymenopteran parasitoids to various potential trap crops: Brassica nigra L., Raphanus sativus var. olifera Pers. and Eruca sativa Mill. with that to B. napus. We recorded their abundance, oviposition preferences and the species composition of the parasitoids.Our results show that oviposition rates of the pollen beetle and its parasitoids as well the species composition of the parasitoids varies with plant species. We discuss the potential of these plant species, especially B. nigra, to enhance the natural control of the beetle by fostering several parasitoid species. The species composition of the parasitoids on different host plants compared with on B. napus is presented for the first time. In addition to trapping pests, the trap crops could also act as parasitoid banks, enhancing natural control of the pest through providing suitable hosts for natural enemies, without increasing the population growth of the next generation of pests.  相似文献   
10.
旨在利用毕赤酵母分泌表达gp96-scFv抗体,纯化后得到能特异性结合gp96抗原的小分子抗体片段(scFv)。根据gp96-scFv抗体基因序列,合成gp96-scFv抗体基因序列,将gp96-scFv抗体序列克隆到毕赤酵母表达质粒pPICZα-A,线性化的重组表达载体电转化到毕赤酵母X33,甲醇诱导目的蛋白表达,通过亲和层析法纯化目的蛋白,并以SDS-PAGE和Western blotting进行鉴定。通过Western blotting、Immunofluorescence、ELISA、FACS方法对gp96-scFv抗体的生物活性进行了检测。结果成功地构建了分泌表达抗gp96蛋白scFv抗体的毕赤酵母菌,每升毕赤酵母菌培养上清经纯化可获约50 mg gp96-scFv抗体,所获抗体其分子量大约为15 kDa,具有与gp96抗原特异性结合的活性。本研究通过毕赤酵母菌成功表达了gp96-scFv抗体,生物活性Western blotting、Immunofluorescence、ELISA、FACS分析表明该抗体能特异性结合gp96。  相似文献   
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