Cold-shock induced high-yield protein production in Escherichia coli

Overexpression of proteins in Escherichia coli at low temperature improves their solubility and stability. Here, we apply the unique features of the cspA gene to develop a series of expression vectors, termed pCold vectors, that drive the high expression of cloned genes upon induction by cold-shock. Several proteins were produced with very high yields, including E. coli EnvZ ATP-binding domain (EnvZ-B) and Xenopus laevis calmodulin (CaM). The pCold vector system can also be used to selectively enrich target proteins with isotopes to study their properties in cell lysates using NMR spectroscopy. We have cloned 38 genes from a range of prokaryotic and eukaryotic organisms into both pCold and pET14 (ref. 3) systems, and found that pCold vectors are highly complementary to the widely used pET vectors.     

Dynamic redistribution of raft domains as an organizing platform for signaling during cell chemotaxis

Spatially restricted activation of signaling molecules governs critical aspects of cell migration; the mechanism by which this is achieved nonetheless remains unknown. Using time-lapse confocal microscopy, we analyzed dynamic redistribution of lipid rafts in chemoattractant-stimulated leukocytes expressing glycosyl phosphatidylinositol-anchored green fluorescent protein (GFP-GPI). Chemoattractants induced persistent GFP-GPI redistribution to the leading edge raft (L raft) and uropod rafts of Jurkat, HL60, and dimethyl sulfoxide-differentiated HL60 cells in a pertussis toxin-sensitive, actin-dependent manner. A transmembrane, nonraft GFP protein was distributed homogeneously in moving cells. A GFP-CCR5 chimera, which partitions in L rafts, accumulated at the leading edge, and CCR5 redistribution coincided with recruitment and activation of phosphatidylinositol-3 kinase gamma in L rafts in polarized, moving cells. Membrane cholesterol depletion impeded raft redistribution and asymmetric recruitment of PI3K to the cell side facing the chemoattractant source. This is the first direct evidence that lipid rafts order spatial signaling in moving mammalian cells, by concentrating the gradient sensing machinery at the leading edge.     

Medicago truncatula Mt-ZFP1 encoding a root enhanced zinc finger protein is regulated by cytokinin, abscisic acid and jasmonate, but not cold.

A Medicago truncatula zinc finger protein cDNA (Mt-ZFP1) was isolated from a M.truncatula seedling cDNA library using RT-PCR product as a probe. The predicted amino acid sequence of Mt-ZFP1 is over 79% similar to S-SCOF-1 from soybean, a novel cold-inducible zinc finger protein involved in cold stress signal transduction mediated by abscisic acid (ABA). The secondary structure of Mt-ZFP1 protein was almost identical to that of S-SCOF-1. Mt-ZFP1 also contained a typical C2H2-type zinc finger domain and a putative nuclear located signal. RNA gel blot hybridization demonstrated that the Mt-ZFP1 gene was actively expressed in roots, with a lower abundance in leaf and stem tissues. Cold treatment did not induce the expression of Mt-ZFP1 in either leaves and stems or roots. Exogenous application of cytokinins marginally increased the accumulation of Mt-ZFP1 mRNA, while ABA and jasmonate treatments decreased the levels of Mt-ZFP1 mRNA. DNA gel-blot analysis demonstrated that Mt-ZFP1 is present as a single copy gene in the M. truncatula genome. These data suggest that Mt-ZFP1 is a novel zinc finger protein with different physiological functions to that of S-SCOF-1. The similar cold-inducible factor like S-SCOF-1 might not exist in M. truncatula.     

Utilization of blueberry by the lappet moth, Streblote panda Hübner (Lepidoptera: Lasiocampidae): survival, development, and larval performance

The lappet moth, Streblote panda Hübner [1820] (Lasiocampidae), is a common species found in blueberry, Vaccinium spp. (Ericaceae) fields of Western Andalusia. The biology of this species as well as the extent to which its larvae can use and survive on blueberry is unknown. In this study, the suitability to larvae of several blueberry cultivars was studied. Larvae were grown under controlled laboratory conditions on excised foliage of six blueberry cultivars. Survival, development, and food use were determined for first and fifth instars. According to our results, blueberry has become an alternative host plant for S. panda in southwestern Andalusia. Low growth rates and efficiencies of use of food were observed. Lower gross efficiency of growth was found for larvae fed blueberry 'Sharpblue', despite a higher apparent digestibility of this cultivar. Larvae reared on this cultivar had the highest mortality, increased developmental time, and used a greater part of metabolism for maintenance. Herbivore pressure may be increased with the widespread planting of the most suitable cultivars 'Misty' and 'O'Neal', whereas 'Sharpblue' and'Climax' seem to be the least suitable host plants. These data provide useful information for planning and managing blueberry orchards in the presence of S. panda populations.     

Subcellullar localization of tumor-associated antigen 3H11Ag

3H11Ag, a tumor-associated antigen defined by the monoclonal antibody 3H11 that specifically recognizes cancer cells in various tumor tissues, was successfully cloned recently, but its function is unknown. To explore the potential roles it plays in tumors, we analyzed its subcellular localization in the present study. By expressing 3H11Ag fused with fluorescent protein in COS-7 cells, we found that 3H11Ag localizes to both cytoplasm and nucleus, which was confirmed by subcellular fractionation. And sequentially extracting the nuclei of COS-7 cells transfected with 3H11Ag showed that it is a DNA- and nuclear matrix-associated protein. Moreover, by expressing a series of red fluorescent protein-tagged truncated forms of 3H11Ag, it was demonstrated that the 150 amino acid residues at its C-terminal are fully responsible for the subcellular localization. In addition, the results of the computational analysis of 3H11Ag were in accordance with those of the experimental analysis. All these data would be helpful to elucidate the functions of 3H11Ag.     

Role of interaction of mannan-binding protein with meprins at the initial step of complement activation in ischemia/reperfusion injury to mouse kidney

Ischemia/reperfusion (I/R) is an important cause of acute renal failure. Recent studies have shown that the complement system mediated by the mannan-binding protein (MBP), which is a C-type serum lectin recognizing mannose, fucose and N-acetylglucosamine residues, plays a critical role in the pathogenesis of ischemic acute renal failure. MBP causes complement activation through the MBP lectin pathway and a resulting complement component, C3b, is accumulated on the brush borders of kidney proximal tubules in a renal I/R-operated mouse kidney. However, the initial step of the complement activation has not been studied extensively. We previously identified both meprins α and β, highly glycosylated zinc metalloproteases, localized on kidney proximal tubules as endogenous MBP ligands. In the present study, we demonstrated that serum-type MBP (S-MBP) and C3b were co-localized with meprins on both the cortex and the medulla in the renal I/R-operated mouse kidney. S-MBP was indicated to interact with meprins in vivo in the I/R-operated mouse kidney and was shown to initiate the complement activation through the interaction with meprins in vitro. Taken together, the present study strongly suggested that the binding of S-MBP to meprins triggers the complement activation through the lectin pathway and may cause the acute renal failure due to I/R on kidney transplantation and hemorrhagic shock.     

Scanning assay of β-galactosidase activity

β-galactosidase, encoded by the lacZ gene in E. coli, can cleave lactose and structurally related compounds to galactose and glucose or structurally related products. Its activity can be measured using an artificial substrate, o-nitrophenyl-β-D-galactopyranoside (ONPG). Miller firstly described the standard quantitative assay of β-galactosidase activity in the cells of bacterial cultures by disrupting the cell membrane with the permeabilization solution instead of preparing cell extracts. Therefore, β-galactosidase became one of the most widely used reporters of gene expression in molecular biology to reflect intracellular gene expression difference. But the Miller assay procedure could not monitor the β-galactosidase reaction in real time and its results were greatly influenced by some operations in the Miller procedure, such as permeabilization time, reaction time and concentration of the cell suspension. A scanning method based on the Miller method to determine the intracellular β-galactosidase activity in E. coli Tuner (DE3) expressing β-galactosidase in real time was developed and the permeabilization time of cells was optimized for that. The comparison of 3 assays of β-galactosidase activity (Miller, colorimetric and scanning) was made. The results proved that scanning method for the determination of enzyme activity with using ONPG as substrate is simple, fast and reproducible.     

Stronger condition dependence in female size explains altitudinal variation in sexual size dimorphism of a Tibetan frog

The present study investigated altitudinal variation in sexual size dimorphism of a Tibetan frog Nanorana parkeri. Size dimorphism was female‐biased in all populations, although this bias became less at higher altitudes because of a steeper altitudinal decrease in female size than male size. Operational sex ratios, an indicator of the opportunity for sexual selection on larger males, changed independently of altitude. Clutch volume, an indicator of the strength of fecundity selection on larger females, was positively with female size, and tended to decrease approaching high altitudes. Females lived longer and grew more slowly than males, and the mean age in both sexes increased and growth rate decreased altitudinally, although the changes were more rapid in females than males. These results suggest that, relative to males, females (i.e. the sex that typically bears greater reproductive costs and experiences stronger directional selection for larger size to take fecundity advantages) should be more sensitive to environments, attaining a larger size via enhancing growth under favourable lower‐latitude conditions but a smaller size as a result of retarding growth when conditions become harsher at higher altitudes. This supports the condition‐dependence hypothesis with respect to intraspecific variation in sexual size dimorphism. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 107 , 558–565.     

基于条形码ITS2序列的青天葵及其混伪品分子鉴别

通过分析青天葵及其常见混伪品的ITS2序列,建立青天葵新型真伪鉴别方法.采用植物基因组DNA提取试剂盒提取青天葵及其混伪品的叶片DNA,一对通用引物PCR扩增ITS2基因片段并直接双向测序.采用DNAMAN、ClustalX软件拼接比对序列,MEGA4.0软件构建NJ树,Schultz等建立的数据库和网站预测ITS2序列的二级结构.结果显示,获得的12条ITS2序列的长度范围为203-242 bp,GC含量范围为53.1％-71.8％.所有样品ITS2序列比对后的长度为249 bp,其中存在226个变异位点.青天葵种间K2P遗传距离(1.125)远大于种内K2P遗传距离(0.004).基于ITS2的序列的NJ树和二级结构均能直观地区分青天葵及其混伪品.