Cell-free synthesis of membrane proteins: Tailored cell models out of microsomes

Incorporation of proteins in biomimetic giant unilamellar vesicles (GUVs) is one of the hallmarks towards cell models in which we strive to obtain a better mechanistic understanding of the manifold cellular processes. The reconstruction of transmembrane proteins, like receptors or channels, into GUVs is a special challenge. This procedure is essential to make these proteins accessible to further functional investigation. Here we describe a strategy combining two approaches: cell-free eukaryotic protein expression for protein integration and GUV formation to prepare biomimetic cell models. The cell-free protein expression system in this study is based on insect lysates, which provide endoplasmic reticulum derived vesicles named microsomes. It enables signal-induced translocation and posttranslational modification of de novo synthesized membrane proteins. Combining these microsomes with synthetic lipids within the electroswelling process allowed for the rapid generation of giant proteo-liposomes of up to 50 μm in diameter. We incorporated various fluorescent protein-labeled membrane proteins into GUVs (the prenylated membrane anchor CAAX, the heparin-binding epithelial growth factor like factor Hb-EGF, the endothelin receptor ETB, the chemokine receptor CXCR4) and thus presented insect microsomes as functional modules for proteo-GUV formation. Single-molecule fluorescence microscopy was applied to detect and further characterize the proteins in the GUV membrane. To extend the options in the tailoring cell models toolbox, we synthesized two different membrane proteins sequentially in the same microsome. Additionally, we introduced biotinylated lipids to specifically immobilize proteo-GUVs on streptavidin-coated surfaces. We envision this achievement as an important first step toward systematic protein studies on technical surfaces.     

Design and selection of trap color for capture of the tea leafhopper,Empoasca vitis,by orthogonal optimization

The tea leafhopper, Empoasca vitis (Göthe) (Hemiptera: Cicadellidae), is a major pest of the tea plant, Camellia sinensis (L.) O. Kuntze (Theaceae). In this study, the RGB color model was used to describe the colors of sticky traps. The most effective color for attraction of E. vitis was investigated by orthogonal optimization. The selected color was verified in tea gardens and the most effective height for positioning of color sticky traps for capturing tea leafhoppers was investigated. After the determination of the effect of the three color parameters and their interactions by orthogonal optimization, the color gold (RGB: 255, 215, 0) was selected as the most effective color to trap tea leafhoppers. In tea gardens, more leafhoppers were captured using gold sticky traps (RGB: 226, 204, 4) than using commercially available yellow sticky traps. The most effective height of gold sticky traps for trapping leafhoppers was 40–60 cm above the tea canopy. Few lady beetles were captured at this height. We conclude that the orthogonal optimization method is a convenient and efficient method to screen digitally generated colors for attracting and trapping of pests.     

Sex ratios and Ophryocystis elektroscirrha infection levels of Danaus plexippus during spring migration through Oklahoma,USA

Monarch butterflies, Danaus plexippus L. (Lepidoptera: Nymphalidae), have a multiple brood migration in the spring as they move between their overwintering grounds and summer breeding grounds. In Oklahoma, USA, monarchs produce at least one generation in the spring, which develops and continues the northward migration, leaving Oklahoma without a breeding population during the hot summer months. Female monarchs leave the overwintering grounds prior to males, but it is not clear whether females re‐colonize areas along the migration route prior to, or at the same time as males. Male‐to‐female ratios are 1:1 at emergence, but studies have identified a male‐biased sex ratio in the field. Both males and females are susceptible to infection by the obligate protozoan parasite, Ophryocystis elektroscirrha McLaughlin & Myers (OE), which reduces flight abilities and life spans of infected individuals. We examine sex ratios during the spring migration through Oklahoma and whether sex ratios or OE infection estimates vary with capture technique (active or passive). Our data suggest populations are male‐biased during the 1st week of spring migration in Oklahoma, but shift to female‐biased by the 3rd week in both cool and warm springs. Therefore, males may leave southern areas prior to females or migrate longer distances per day. Active sampling (i.e., netting) did not bias sex compared to passive sampling (i.e., sticky traps). Significantly fewer OE‐carrying monarchs (with two or more spores) were captured via netting than by sticky traps which may be caused by sticky trap glue affecting tape sampling effectiveness, but there was no difference in the number of heavily infected individuals (more than 100 spores). Therefore, data from netted monarchs may underestimate OE infection rates within populations.     

微生物黄色素的研究进展

对来源于微生物的天然黄色素的研究进行了详细概述, 尤其对红曲黄色素的生产、安全性以及黄色素的合成代谢机理研究进行了详细论述, 最后对红曲黄色素的应用前景及其未来研究重点进行了展望, 并对研究中存在的问题进行了分析。     

Vertical oviposition and Lambornella clarki (Ciliophora: Tetrahymenidae) dispersal by Aedes sierrensis (Diptera: Culicidae) in California

Vertical stratification of treeholes used by mosquitoes may reflect resource quality or result from interspecific competition. Mosquitoes able to monopolize treeholes with optimal resources may be over‐represented in the community. Aedes sierrensis, which is well adapted for the Mediterranean climate of California, has evolved in the absence of interspecific competition, so oviposition should reflect resource quality to a large extent. Artificial oviposition traps mounted at four canopy heights facing north or south on trees in a mixed‐oak forest at four elevations of the Pacific Coastal Range were used to assess vertical ovipositional preferences by the western treehole mosquito. Natural dispersal of the ciliated protozoan parasite Lambornella clarki was similarly monitored. Gravid Ae. sierrensis showed no vertical stratification during egg laying in traps. Lambornella clarki were naturally dispersed at relatively low frequency into traps and persisted unless eliminated by larval predation. Aedes sierrensis is not currently constrained into occupying a subset of treeholes. However, invasion of its native range by competitive species may alter oviposition patterns.     

Zucchini Yellow Fleck Virus is an Emergent Virus on Melon in Sicily (Italy)

Since 2006, winter melon plants (Cucumis melo L. var inodorus) showing symptoms of pin‐point yellow spots were noticed in Sicily (Italy). Leaf samples were tested by enzyme‐linked immunosorbent assay to the most important viruses‐infecting cucurbits. Zucchini yellow fleck virus (ZYFV, genus Potyvirus) was the only virus detected. Surveys in 2007 and 2008 revealed an increasing number of sites in Sicily with ZYFV‐infected winter melon plants. To confirm the identity of the virus as ZYFV, two isolates from different locations were sequenced and shown to be approximately 85% identical to the published sequences of isolates previously identified in Italy and France. This is the first report of ZYFV occurring on melon in Italy.     

Differential thermal sensitivity between the recipient ooplasm and the donor nucleus in Holstein and Taiwan native yellow cattle

The objective of this study was to compare thermal sensitivity of recipient ooplasm and donor nucleus from Holstein and Taiwan native yellow (TY) cows. Oocytes and cumulus cells from each breed were incubated at 43 °C (heat shock) or 38.5 °C (control) for 1 h prior to nucleus transplantation. Reconstructed embryos cloned by transfer of non-heated Holstein donor cells to heat-shocked Holstein ooplasm (Ho⁺-Hd⁻) had a lower (P < 0.05) blastocyst rate than those cloned from non-heated Holstein ooplasm receiving heated (Ho⁻-Hd⁺) or non-heated (Ho⁻-Hd⁻) Holstein donor cells (11.3 vs. 34.3 or 36.8%). Heat-shocked donor cells from either Holstein or TY cows did not significantly affect blastocyst rates of reconstructed embryos produced from Holstein ooplasm (30.6-32.9%). In contrast, blastocyst rates of reconstructed embryos generated with heat-shocked Holstein ooplasm were lower (P < 0.05) than that with heat-shocked TY ooplasm (11.2 vs 45.2%). Without heat shock, embryos reconstructed by transferring donor cells to ooplasm of Holstein or TY cows had similar (P > 0.05) blastocyst rates (28.9-33.3%). Transplantation of reconstructed embryos (n = 30) to recipients (n = 23) resulted in three live calves, derived from embryos cloned with TY ooplasm and donor nuclei from either Holstein (n = 2) or TY cows (n = 1). In conclusion, ooplasm of TY cattle was more resistant to heat stress than that derived from Holsteins; therefore, ooplasm may be a major determinant for thermal sensitivity in bovine oocytes and embryos.     

Biology of the solitary wasp Trypoxylon (Trypargilum) agamemnon Richards 1934 (Hymenoptera: Crabronidae) in trap‐nests

Buschini, M.L.T. and Fajardo, S. 2009. Biology of the solitary wasp Trypoxylon (Trypargilum) agamemnon Richards 1934 (Hymenoptera: Crabronidae) in trap‐nests. —Acta Zoologica (Stockholm) 91 : 426–432. Some Trypoxylon species build their nests in preexisting tubular cavities like hollow stems and beetle borings in branches. Study of the biology of these insects is relatively easy because the females of these wasps nest with enormous success in trap‐nests. The aim of this study was to investigate the abundance, seasonality and life‐history of Trypoxylon agamemnon. For capture of these insects, trap‐nests were installed in the Parque Municipal das Araucárias in araucaria forest, grassland and swamp, from December, 2001 to December, 2005. Two hundred and ninety seven nests were obtained. They were constructed more often during the summer (from December to April). The nests were built only in araucaria forest and consisted of a linear series of cells, divided by mud partitions, whose number varied from 1 to 7. Normally they have only one vestibular cell. The inner cells had been provisioned, usually with spiders of Anyphaenidae family. Sex‐ratio was strongly female biased. Its main natural enemies included Chrysididae, Ichneumonidae and Tachinidae.     

Hemagglutinin of Influenza Virus Partitions into the Nonraft Domain of Model Membranes

The HA of influenza virus is a paradigm for a transmembrane protein thought to be associated with membrane-rafts, liquid-ordered like nanodomains of the plasma membrane enriched in cholesterol, glycosphingolipids, and saturated phospholipids. Due to their submicron size in cells, rafts can not be visualized directly and raft-association of HA was hitherto analyzed by indirect methods. In this study, we have used GUVs and GPMVs, showing liquid disordered and liquid ordered domains, to directly visualize partition of HA by fluorescence microscopy. We show that HA is exclusively (GUVs) or predominantly (GPMVs) present in the liquid disordered domain, regardless of whether authentic HA or domains containing its raft targeting signals were reconstituted into model membranes. The preferential partition of HA into ld domains and the difference between lo partition in GUV and GPMV are discussed with respect to differences in packaging of lipids in membranes of model systems and living cells suggesting that physical properties of lipid domains in biological membranes are tightly regulated by protein-lipid interactions.     

黄热病病毒检测微阵列的研究制备

目的:制备黄热病病毒寡核苷酸检测微阵列.方法:根据黄热病病毒基因组序列,并应用生物信息学软件设计出寡核苷酸探针用于制备基因芯片,克隆于质粒上的黄热病病毒全长基因DNA经限制性显示技术扩增并标记,完成杂交后对芯片进行扫描和数据分析.结论:微阵列检测技术为检测黄热病病毒提供了一种早期、快速、可靠的方法,具有应用于临床检测的前景.