Using chimeric piggyBac transposase to achieve directed interplasmid transposition in silkworm Bombyx mori and fruit fly Drosophila cells

The piggyBac transposon has been long used to integrate foreign DNA into insect genomes. However, undesirable transgene expression can result from random insertions into the genome. In this study, the efficiency of chimeric Gal4-piggyBac transposase in directing integration onto a DNA target plasmid was evaluated in cultured silkworm Bombyx mori Bm-12 and fruit fly Drosophila Schneider 2 (S2) cells. The Gal4-piggyBac transposase has a Gal4 DNA-binding domain (DBD), and the target plasmid has upstream activating sequences (UAS) to which the Gal4 DBD can bind with high affinity. The results indicate that, in the Bm-12 and S2 cells, transpositional activity of Gal4-piggyBac transposase was increased by 4.0 and 7.5 times, respectively, compared to controls, where Gal4-UAS interaction was absent. Moreover, the Gal4-piggyBac transposase had the ability of directing piggyBac element integration to certain sites of the target plasmid, although the target-directing specificity was not as high as expected. The chimeric piggyBac transposase has the potential for use in site-directed transgenesis and gene function research in B. mori.     

Caffeic acid product from the highly copper-tolerant plant Elsholtzia splendens post-phytoremediation: its extraction, purification, and identification

In the current study, caffeic acid was an important metabolite in the highly copper-tolerant plant Elsholtzia splendens. Preparation and purification of caffeic acid were performed on the dried biomass of the plants by means of sonication/ethanol extraction, followed by purification using a macroporous resin (D101 type) column and silica gel chromatography. The faint-yellow caffeic acid product was yielded with a purity of 98.46%, and it was chemically identified from spectra of Fourier transform infrared spectroscopy (FTIR), proton nuclear magnetic resonance (¹H NMR)/carbon nuclear magnetic resonance (¹³C NMR), and electrospray ionization mass spectrometry (ESI-MS). Caffeic acid is a possible product from the post-harvest processing of Elsholtzia splendens biomass.     

Genetic variation in alkaloid accumulation in leaves of Nicotiana

Alkaloids are plant secondary metabolites that are widely distributed in Nicotiana species and contribute greatly to the quality of tobacco leaves. Some alkaloids, such as nornicotine and myosmine, have adverse effects on human health. To reduce the content of harmful alkaloids in tobacco leaves through conventional breeding, a genetic study of the alkaloid variation among different genotypes is required. In this study, alkaloid profiles in leaves of five Nicotiana tabacum cultivars and Nicotiana tomentosiformis were investigated. Six alkaloids were identified from all six genotypes via gas chromatograph-mass spectrometry (GC-MS). Significant differences in alkaloid content were observed both among different leaf positions and among cultivars. The contents of nornicotine and myosmine were positively and significantly correlated (R ²=0.881), and were also separated from those of other alkaloids by clustering. Thus, the genotype plays a major role in alkaloid accumulation, indicating a high potential for manipulation of alkaloid content through traditional breeding.     

题目：遗传改造细菌基因组的策略：基因定点突变、基因失活和基因过表达

With the availability of the whole genome sequence of Escherichia coli or Corynebacterium glutamicum, strategies for directed DNA manipulation have developed rapidly. DNA manipulation plays an important role in understanding the function of genes and in constructing novel engineering bacteria according to requirement. DNA manipulation involves modifying the autologous genes and expressing the heterogenous genes. Two alternative approaches, using electroporation linear DNA or recombinant suicide plasmid, allow a wide variety of DNA manipulation. However, the over-expression of the desired gene is generally executed via plasmid-mediation. The current review summarizes the common strategies used for genetically modifying E. coli and C. glutamicum genomes, and discusses the technical problem of multi-layered DNA manipulation. Strategies for gene over-expression via integrating into genome are proposed. This review is intended to be an accessible introduction to DNA manipulation within the bacterial genome for novices and a source of the latest experimental information for experienced investigators.     

Protective effect of DNA-mediated immunization with liposome-encapsulated GRA4 against infection of Toxoplasma gondii

The dense granule protein 4 (GRA4) is a granular protein from Toxoplasma gondii, and is a candidate for vaccination against this parasite. In this study, the plasmid pcDNA3.1-GRA4 (pGRA4), encoding for the GRA4 antigen, was incorporated by the dehydration-rehydration method into liposomes composed of 16 mmol/L egg phosphatidylcholine (PC), 8 mmol/L dioleoyl phosphatidylethanolamine (DOPE), and 4 mmol/L 1,2-diodeoyl-3-(trimethylammonium) propane (DOTAP). C57BL/6 mice and BALB/c mice were immunized intramuscularly three times with liposome-encapsulated pGRA4 to determine whether DNA immunization could elicit a protective immune response to T. gondii. Enzyme-linked immunosorbent assay (ELISA) of sera from immunized mice showed that liposome-encapsulated pGRA4 generated high levels of IgG antibodies to GRA4. Production of primary interferon (IFN)-γ and interleukin (IL)-2 in GRA4-stimulated splenocytes from vaccinated mice suggested a modulated Th1-type response. 72.7% of C57BL/6 mice immunized with liposome-encapsulated pGRA4 survived the challenge with 80 tissue cysts of ME49 strain, whereas C57BL/6 mice immunized with pGRA4 had only a survival rate of 54.5%. When immunized BALB/c mice were intraperitoneally challenged with 10³ tachyzoites of the highly virulent RH strain, the survival time of mice immunized with liposome-encapsulated pGRA4 was markedly longer than that of other groups. Our observations show that liposome-encapsulated pGRA4 enhanced the protective effect against infection of T. gondii. Project supported by the Science Foundation of the Health Bureau of Zhejiang Province, China (Nos. 2003QN003 and 2005A001) and the Science Foundation of the Science and Technology Department of Zhejiang Province, China (No. 2006C13022)     

Enhancing cellular immune response to HBV M DNA vaccine in mice by codelivery of interleukin-18 recombinant

Objective: To investigate the effect of interleukin-18 (IL-18) on immune response induced by plasmid encoding hepatitis B virus middle protein antigen and to explore new strategies for prophylactic and therapeutic HBV DNA vaccines. Methods: BALB/c mice were immunized with pCMV-M alone or co-immunized with pcDNA3-18 and pCMV-M and then their sera were collected for analysing anti-HBsAg antibody by ELISA; splenocytes were isolated for detecting specific CTL response and cytokine assay in vitro. Results: The anti-HBs antibody level of mice co-immunized with pcDNA3-18 and pCMV-M was slightly higher than that of mice immunized with pCMV-M alone, but there was not significantly different (P>0.05). Compared with mice injected with pCMV-M, the specific CTL cytotoxity activity of mice immunized with pcDNA3-18 and pCMV-M was significantly enhanced (P<0.05) and the level of IFN-γ in supernatant of splenocytes cultured with HBsAg in vitro was significantly elevated (P<0.05) while the level of IL-4 had no significant difference (P>0.05). Conclusion: The plasmid encoding IL-18 together with HBV M gene DNA vaccines may enhance specific TH1 cells and CTL cellular immune response induced in mice, so that IL-18 is a promising immune adjuvant.     

Enhancing cellular immune response to HBV M DNA vaccine in mice by codelivery of interleukin-18 recombinant

INTRODUCTIONChronicinfectionwithHBVaffectsmorethan250millionpeopleworldwide.Therearemorethan120millionchronicHBVcarriersinChina;appro-ximately10percentofthemremaininstateofchronichepatitisandhaveahighriskofdevelop-mentofcirrhosisandhepatocellularcarcinoma.ButthereisnoeffectivemethodtocontrolchronicHBVinfectionatpresent.Recentdataindicatedthatim-munotherapeuticstrategiesstimulatingbothcellularandhumoralimmuneresponsestoHBVantigensareessentialforcuringchronicHBVinfection(ChisariandFe…     

Comprehensive screening and selection of okra (Abelmoschus esculentus) germplasm for salinity tolerance at the seedling stage and during plant ontogeny

The okra germplasm was screened for salinity tolerance at the seedling stage and during plant ontogeny. Substantial variation existed in okra for salinity tolerance at the seedling stage. An 80 mmol/L NaCl concentration was suitable for discriminating tolerant and non-tolerant okra genotypes. The pooled ranking of the genotypes, based on individual rankings for each trait (root and shoot length, germination percentage, and relative Na⁺ and K⁺) in individual NaCl concentrations, was effective for selecting tolerant genotypes. Genotypes selected at the seedling stage maintained their tolerance to NaCl during plant ontogeny, suggesting that screening of the germplasm entries and advanced breeding materials for salt tolerance at the seedling stage is effective. Among 39 okra genotypes, five were identified as the most tolerant genotypes and showed potential for use in breeding programs that focus on the development of salt-tolerant, high-yield okra cultivars.     

Mass spectrometry-based protein-protein interaction techniques and their applications in studies of DNA damage repair

Proteins are major functional units that are tightly connected to form complex and dynamic networks.These networks enable cells and organisms to operate properly and respond efficiently to environmental cues.Over the past decades,many biochemical methods have been developed to search for protein-binding partners in order to understand how protein networks are constructed and connected.At the same time,rapid development in proteomics and mass spectrometry(MS)techniques makes it possible to identify interacting proteins and build comprehensive protein-protein interaction networks.The resulting interactomes and networks have proven informative in the investigation of biological functions,such as in the field of DNA damage repair.In recent years,a number of proteins involved in DNA damage response and DNA repair pathways have been uncovered with MS-based protein-protein interaction studies.As the technologies for enriching associated proteins and MS become more sophisticated,the studies of protein-protein interactions are entering a new era.In this review,we summarize the strategies and recent developments for exploring protein-protein interaction.In addition,we discuss the application of these tools in the investigation of protein-protein interaction networks involved in DNA damage response and DNA repair.     

Effects of medium composition on the production of plasmid DNA vector potentially for human gene therapy

INTRODUCTION The difficulties associated with large-scaleproduction of biotherapeutics provide a constantchallenge to the biotechnology industry. FDA hadadded “therapeutic DNA plasmid vectors” to the listof well-characterized biotechnology product (DoHHs,1996), and gene therapy has moved rapidly fromlaboratory scale to clinical trials. It is urgent to de-velop new protocols to obtain high-quality plasmidswith high yields and minimal or no contamination ofRNA and chromosomal D…     

Manufacturer identification and storage time determination of “Dong’e Ejiao” using near infrared spectroscopy and chemometrics

We have developed a set of chemometric methods to address two critical issues in quality control of a precious traditional Chinese medicine (TCM), Dong’e Ejiao (DEEJ). Based on near infrared (NIR) spectra of multiple samples, the genuine manufacturer of DEEJ, e.g. Dong’e Ejiao Co., Ltd., was accurately identified among 21 suppliers by the fingerprint method using Hotelling T², distance to Model X (DModX), and similarity match value (SMV) as discriminate criteria. Soft independent modeling of the class analogy algorithm led to a misjudgment ratio of 6.2%, suggesting that the fingerprint method is more suitable for manufacturer identification. For another important feature related to clinical efficacy of DEEJ, storage time, the partial least squares-discriminant analysis (PLS-DA) method was applied with a satisfactory misjudgment ratio (15.6%) and individual prediction error around 1 year. Our results demonstrate that NIR spectra comprehensively reflect the essential quality information of DEEJ, and with the aid of proper chemometric algorithms, it is able to identify genuine manufacturer and determine accurate storage time. The overall results indicate the promising potential of NIR spectroscopy as an effective quality control tool for DEEJ and other precious TCM products.     

Analysis of 19-nortestosterone residue in animal tissues by ion-trap gas chromatography-tandem mass spectrometry

A rapid sample treatment procedure for the gas chromatography-tandem mass spectrometry (GC-MS) determination of 19-nortestosterone (19-NT) in animal tissues has been developed. In our optimized procedures, enzymatic hydrolysis with β-glucuronidase from Escherichia coli was performed in an acetate buffer (pH 5.2, 0.2 mol/L). Next, the homogenate was mixed with methanol and heated at 60 °C for 15 min, then placed in an ice-bath at −18 °C for 2 h. After liquid-liquid extraction with n-hexane, the analytes were subjected to a normal-phase solid phase extraction (SPE) C₁₈ cartridge for clean-up. The dried organic extracts were derivatized with heptafluorobutyric anhydride (HFBA), and then the products were injected into GC-MS. Using electron impact mass spectrometry (EI-MS) with positive chemical ionization (PCI), four diagnostic ions (m/z 666, 453, 318, and 306) were determined. A standard calibration curve over the concentration range of 1–20 ng/g was reached, with Y=467 084X-68 354 (R ²=0.999 7) for 19-NT, and the detection limit was 0.3 ng. When applied to spiked samples collected from bovine and ovine, the recoveries ranged from 63% to 101% with relative standard deviation (RSD) between 2.7% and 8.9%. The procedure is a highly efficient, sensitive, and more economical method which offers considerable potential to resolve cases of suspected nandrolone doping in husbandry animals.     

Transgenic Brassica chinensis plants expressing a bacterial codA gene exhibit enhanced tolerance to extreme temperature and high salinity

Transgenic Brassica compestris L.spp.chinensis plants expressing a choline oxidase(codA) gene from Arthrobacter globiformis were obtained through Agrobacterium tumefaciens-mediated transformation.In the transgenic plants,codA gene expression and its product transportation to chloroplasts were detected by the enzyme-linked immunosorbent assay(ELISA) examination,immunogold localization,and 1 H-nuclear magnetic resonance( 1 H-NMR) . Stress tolerance was evaluated in the T3 plants under extreme temperature and salinity conditions.The plants of transgenic line 1(L1) showed significantly higher net photosynthetic rate(Pn) and Pn recovery rate under high(45°C,4 h) and low temperature(1°C,48 h) treatments,and higher photosynthetic rate under high salinity conditions(100,200,and 300 mmol/L NaCl,respectively) than the wild-type plants.The enhanced tolerance to high temperature and high salinity stresses in transgenic plants is associated with the accumulation of betaine,which is not found in the wild-type plants.Our results indicate that the introduction of codA gene from Arthrobacter globiformis into Brassica compestris L.spp.chinensis could be a potential strategy for improving the plant tolerance to multiple stresses.     

Effect of lipopolysaccharide on the hemocyte apoptosis of Eriocheir sinensis

In the present study, we investigated the possible toxicity mechanism of lipopolysaccharide (LPS) extracted from Gram-negative bacteria in Eriocheir sinensis hemocytes. Apoptotic hemocytes and reactive oxygen species (ROS) production induced by the LPS were monitored by the combination of flow cytometry and microscope observation. It was shown that LPS induced serious damage on the DNA and morphological changes in hemocytes, including cell shrinkage, fracture of nucleus membrane, margination, condensation and fragmentation of chromatin, and formation of apoptotic bodies indicating obvious hemocyte apoptosis. As compared with the control group, the apoptotic cell ratio increased to 30.61% and 39.01% after 1-h exposure and 57.72% and 75.01% after 2-h exposure to 1 and 10 μg/ml LPS, respectively (P<0.05). Significant outburst of ROS production was observed in LPS-treated hemocytes with approximately 176.6% of relative dichlorofluorescein mean fluorescence at 1-h exposure, followed by a drastic decline (P<0.05). These results indicated that LPS would induce oxidative stress on hemocytes from E. sinensis and cause ROS burst, DNA damage, and subsequently apoptosis. The process of ROS-mediated apoptosis might be one of the potential toxicity mechanisms of LPS on crustacean hemocytes.     

An easy-to-use site-directed mutagenesis method with a designed restriction site for convenient and reliable mutant screening

Site-directed mutagenesis (SDM) has been a very important method to probe the function-structure relationship of proteins. In this study, we introduced an easy-to-use, polymerase chain reaction (PCR)-based SDM method for double-stranded plasmid DNA, with a designed restriction site to ensure simple and efficient mutant screening. The DNA sequence to be mutated was first translated into amino acid sequence and then the amino acid sequence was reversely translated into DNA sequence with degenerate codons, resulting in a large number of sequences with silent mutations, which contained various restriction endonuclease (RE) sites. Certain mutated sequence with an appropriate RE site was selected as the target DNA sequence for designing a pair of mutation primers to amplify the full-length plasmid via inverse PCR. The amplified product was 5'-phosphorylated, circularized, and transformed into an Escherichia coli host. The transformants were screened by digesting with the designed RE. This protocol uses only one pair of primers and only one PCR is conducted, without the need for hybridization with hazardous isotope for mutant screening or subcloning step.     

Cellular mechanism of cardiac alternans: an unresolved chicken or egg problem简

T-wave alternans, a specific form of cardiac alternans, has been associated with the increased susceptibility to cardiac arrhythmias and sudden cardiac death (SCD). Plenty of evidence has related cardiac alternans at the tissue level to the instability of voltage kinetics or Ca²⁺ handling dynamics at the cellular level. However, to date, none of the existing experiments could identify the exact cellular mechanism of cardiac alternans due to the bi-directional coupling between voltage kinetics and Ca²⁺ handling dynamics. Either of these systems could be the origin of alternans and the other follows as a secondary change, therefore making the cellular mechanism of alternans a difficult chicken or egg problem. In this context, theoretical analysis combined with experimental techniques provides a possibility to explore this problem. In this review, we will summarize the experimental and theoretical advances in understanding the cellular mechanism of alternans. We focus on the roles of action potential duration (APD) restitution and Ca²⁺ handling dynamics in the genesis of alternans and show how the theoretical analysis combined with experimental techniques has provided us a new insight into the cellular mechanism of alternans. We also discuss the possible reasons of increased propensity for alternans in heart failure (HF) and the new possible therapeutic targets. Finally, according to the level of electrophysiological recording techniques and theoretical strategies, we list some critical experimental or theoretical challenges which may help to determine the origin of alternans and to find more effective therapeutic targets in the future.     

Immobilization of foreign protein into polyhedra of Bombyx mori nucleopolyhedrovirus (BmNPV)

In the late phase of Bombyx mori nucleopolyhedrovirus (BmNPV) infection, a large amount of polyhedra appear in the infected cell nucleolus, these polyhedra being dense protein crystals protecting the incorporated virions from the harsh environment. To investigate whether the foreign protein could be immobilized into the polyhedra of BmNPV, two recombinant baculoviruses were generated by a novel BmNPV polyhedrin-plus (polh⁺) Bac-to-Bac system, designated as vBmBac(polh⁺)-enhanced green fluorescent protein (EGFP) and vBmBac(polh⁺)-LacZ, which can express the polyhedrin and foreign protein simultaneously. Light microscopy analysis showed that all viruses produced polyhedra of normal appearance. Green fluorescence can be apparently detected on the surface of the vBmBac(polh⁺)-EGFP polyhedra, but not the BmNPV polyhedra. Fluorescence analysis and anti-desiccation testing confirmed that EGFP was embedded in the polyhedra. As expected, the vBmBac(polh⁺)-LacZ polyhedra contained an amount of LacZ and had a higher β-galactosidase activity. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting were also performed to verify if the foreign proteins were immobilized into polyhedra. This study provides a new inspiration for efficient preservation of useful proteins and development of new pesticides with toxic proteins.     

重组质粒pRSET DNA纯化方法的比较

分别采用柱式小量质粒抽提纯化试剂盒和聚乙二醇沉淀法纯化重组质粒pRSET DNA,琼脂糖凝胶电泳和DU800核酸蛋白分析仪鉴定已纯化的重组质粒pRSET DNA的纯度.结果显示,采用聚乙二醇沉淀法纯化重组质粒pRSET DNA:A260/280=1.83,试剂盒所得结果:A260/280=1.91.与试剂盒相比,聚乙二醇沉淀法获得纯化质粒的纯度没有显著性差异,常规质粒纯化实验中可用此法代替昂贵的试剂盒.