三种禽病毒抗原的串联表达、纯化及活性鉴定

为实现多个基因在同一菌株中均一可溶性表达,简化基因工程亚单位多联多价疫苗中抗原生产的工艺步骤,本研究选用Ⅰ群4型禽腺病毒(FAdV-4) Fiber-2蛋白、鸡传染性法氏囊病病毒(IBDV) VP2蛋白和减蛋综合征病毒(EDSV)Fiber蛋白3种来自不同禽病毒的抗原为研究对象,利用原核表达系统,通过密码子优化、载体启动子改造和基因串联顺序优化,获得单一载体/多重转录单元的共表达重组质粒。将共表达重组质粒转化大肠杆菌BL21(DE3)菌株,进行3个基因的共表达。纯化后的蛋白进行Western blotting和蛋白活性检测。结果表明,目的基因经过密码子优化、载体启动子改造和基因串联顺序的优化后,获得均一可溶性共表达的3种蛋白,纯化后蛋白纯度大于80%,Western blotting分析和琼脂扩散试验表明串联表达的3种蛋白具有免疫反应性和抗原活性。文中通过目的基因密码子优化、表达载体启动子改造和基因串联等关键技术的突破,首次实现了3种不同禽病毒抗原的高效、均一、可溶性串联表达和纯化,为基因工程亚单位多联多价疫苗的研制奠定了基础。     

胞外三磷酸腺苷通过一氧化氮调节镉诱导的氧化压力和细胞死亡

采用液体悬浮培养方法,研究胞外三磷酸腺苷(ATP)通过一氧化碳(NO)调节镉诱导对烟草(Nicotiana tabacum L.)悬浮细胞氧化压力和死亡水平的影响。结果显示,镉离子(Cd^2+)可以以剂量依赖的模式引起烟草悬浮细胞氧化压力和死亡水平的上升,而施加外源ATP可有效缓解Cd^2+诱导的氧化压力和细胞死亡。进一步研究发现,和外源ATP的缓解作用相似,NO的供体硝普钠(SNP)同样可以缓解Cd^2+诱导的氧化压力和细胞死亡水平的上升;且NO合成抑制剂(L-NAME)可部分解除外源ATP的缓解作用。研究结果表明外源ATP可通过NO调节镉诱导的氧化压力和细胞死亡。     

Increased adiposity in DNA binding-dependent androgen receptor knockout male mice associated with decreased voluntary activity and not insulin resistance

In men, as testosterone levels decrease, fat mass increases and muscle mass decreases. Increased fat mass in men, in particular central obesity, is a major risk factor for type 2 diabetes, cardiovascular disease, and all-cause mortality. Testosterone treatment has been shown to decrease fat mass and increase fat-free mass. We hypothesize that androgens act directly via the DNA binding-dependent actions of the androgen receptor (AR) to regulate genes controlling fat mass and metabolism. The aim of this study was to determine the effect of a global DNA binding-dependent (DBD) AR knockout (DBD-ARKO) on the metabolic phenotype in male mice by measuring body mass, fat mass, food intake, voluntary physical activity, resting energy expenditure, substrate oxidation rates, serum glucose, insulin, lipid, and hormone levels, and metabolic gene expression levels and second messenger protein levels. DBD-ARKO males have increased adiposity despite a decreased total body mass compared with wild-type (WT) males. DBD-ARKO males showed reduced voluntary activity, decreased food intake, increased serum leptin and adiponectin levels, an altered lipid metabolism gene profile, and increased phosphorylated CREB levels compared with WT males. This study demonstrates that androgens acting via the DNA binding-dependent actions of the AR regulate fat mass and metabolism in males and that the increased adiposity in DBD-ARKO male mice is associated with decreased voluntary activity, hyperleptinemia and hyperadiponectinemia and not with insulin resistance, increased food intake, or decreased resting energy expenditure.     

Responses in the physiology and biochemistry of Korean pine (Pinus koraiensis) under supplementary UV-B radiation

The effect of supplementary UV-B radiation on Korean pine (Pinus koraiensis Sieb. et Zucc) was investigated. Compared with the control, the T1, T2, and T3 UV-B treatments increased by 1.40, 2.81, and 4.22 kJ m^?2 d^?1, respectively. Gas-exchange parameters, photosynthetic pigment concentrations, contents of secondary metabolites, epicuticular wax, free radical, malondialdehyde (MDA), and the activities of antioxidant enzymes were determined after 40 d of exposure. The concentrations of chlorophyll (Chl) a, Chl b, total Chl, carotenoid (Car), and the ratio Chl a/b in the pine needles were in the following order: T1 > T2 > T3. Compared with the control, the contents of flavonoids and epicuticular wax significantly decreased in all levels of supplementary UV-B radiations (p<0.05). Moreover, the contents of hydrogen peroxide (H₂O₂) and MDA significantly increased with the enhanced UV-B radiations (p<0.05). Korean pine can increase the catalase, ascorbate peroxidase, and superoxide dismutase activities to prevent oxidative stress by supplementary UV-B radiation. However, its defence mechanism is not efficient enough to prevent UV-Binduced damage.     

Two novel SNPs of the ABCG2 gene and its associations with milk traits in Chinese Holsteins

The ATP-binding cassette transporter ABCG2 (also known as breast cancer resistance protein, BCRP) belongs to the ATP-binding cassette (ABC) family of transmembrane drug transporters, playing a crucial role in the protection of various cells and tissues against xenotoxins and/or endotoxins. Recently, several studies have proposed it as the potential gene underlying the QTL on bovine chromosome 6. Hence, in this study, the PCR-SSCP method was applied to detect two polymorphisms (A → C and A → G) in the target sequence coding nucleotide-binding domain (NBD) region of ABCG2 and evaluate its associations with milk production traits and mastitis-related traits among Chinese Holsteins. In the analyzed population, the allelic frequencies for the A and B alleles were 0.5990 and 0.4010, respectively and the genotypic frequencies were in Hardy–Weinberg disequilibrium (P < 0.01). Moreover, significant statistical relationships between the polymorphisms of ABCG2 gene and following traits, including milk yields, milk protein percentage and somatic cell scores (SCS), were found (P < 0.05). When compared with AA genotype, BB genotype was associated with higher milk yields during 1st and 2nd lactations, as well as lower milk protein percentage and SCS. Thus, BB genotype is suggested to be a molecular marker for superior milk performance.     

Extracellular hydrolase enzyme production by soil fungi from King George Island, Antarctica

Various microbial groups are well known to produce a range of extracellular enzymes and other secondary metabolites. However, the occurrence and importance of investment in such activities have received relatively limited attention in studies of Antarctic soil microbiota. In order to examine extracellular enzyme production in this chronically low-temperature environment, fungi were isolated from ornithogenic, pristine and human-impacted soils collected from the Fildes Peninsula, King George Island, Antarctica during the austral summer in February 2007. Twenty-eight isolates of psychrophilic and psychrotolerant fungi were obtained and screened at a culture temperature of 4°C for activity of extracellular hydrolase enzymes (amylase, cellulase, protease), using R2A agar plates supplemented with (a) starch for amylase activity, (b) carboxymethyl cellulose and trypan blue for cellulase activity or (c) skim milk for protease activity. Sixteen isolates showed activity for amylase, 23 for cellulase and 21 for protease. One isolate showed significant activity across all three enzyme types, and a further 10 isolates showed significant activity for at least two of the enzymes. No clear associations were apparent between the fungal taxa isolated and the type of source soil, or in the balance of production of different extracellular enzymes between the different soil habitats sampled. Investment in extracellular enzyme production is clearly an important element of the survival strategy of these fungi in maritime Antarctic soils.     

Structure characterization and identification steroidal saponins from Ophiopogon japonicus Ker-Gawler (Liliaceae) by high-performance liquid chromatography with ion trap mass spectrometry

Introduction – Steroidal saponins are the main active constituents in Ophiopogon japonicus Ker‐Gawler (Liliaceae). However, because of their high polarity, non‐chromophores and low content in plants, steroidal saponins are difficult to be isolated from O. japonicus by conventional phytochemical methods. Objective – To develop a sensitive and rapid approach towards the structural analysis of steroidal saponins using HPLC/ESI‐MSⁿ. Methodology – The fragmentation behaviors of six known steroidal saponins in negative ESI‐MSⁿ were used to deduce their mass spectral fragmentation mechanisms. By using HPLC/ESI‐MSⁿ, the important structural information on aglycone types, sugar types and saccharide sequences can be obtained. Results – According to the HPLC retention behaviour, the molecular structural characteristics provided by multistage mass spectrometry spectra and the literature, a total of 8 steroidal saponins were tentatively identified or characterized in O. japonicus rapidly. Conclusion – This work has shown that HPLC‐ESI‐MSⁿ may be used as an effective and rapid method for the characterization and identification of steroidal saponins from O. japonicus. Copyright © 2010 John Wiley & Sons, Ltd.     

Improvement of strain discrimination by combination of RAPD with PFGE for the analysis of the swine isolates of Salmonella enterica serovar Choleraesuis

Salmonella enterica serovar Choleraesuis (S. Choleraesuis) can cause salmonellosis in pigs and humans. Currently, the most common method used for the subtyping of this Salmonella serovar is pulsed field gel electrophoresis (PFGE) using XbaI as a DNA digestion enzyme. In this study, we compared and combined PFGE with the randomly amplified polymorphic DNA method, for the typing of 95 S. Choloraesuis strains isolated from diseased pigs. Using PFGE with XbaI, with AvrII, and with SpeI digested DNA, 29, 74, and 40 patterns, respectively, were obtained. Also, 53, 15, and 35 strains, respectively, belong to the major patterns X1, A1, and S1. When these three digestion patterns were combined, 83 PFGE pattern combinations were obtained. On the other hand, using RAPD with selected primer alone generated 76 patterns, and 11 strains which fell within a single X1A1S1 PFGE combination pattern were discriminated into 10 patterns. Thus, for S. Choloraesuis, PFGE with AvrII allowed higher discrimination than PFGE with XbaI, and some of the PFGE groupings obtained by combining the XbaI, AvrII and SpeI digestion patterns were further subdivided by the RAPD method.     

Structural aspects of binding of α-linked digalactosides to human galectin-1

By definition, adhesion/growth-regulatory galectins are known for their ability to bind β-galactosides such as Galβ(1 → 4)Glc (lactose). Indications for affinity of human galectin-1 to α-linked digalactosides pose questions on the interaction profile with such bound ligands and selection of the galactose moiety for CH-π stacking. These issues are resolved by a combination of (15)N-(1)H heteronuclear single quantum coherence (HSQC) chemical shift and saturation transfer difference nuclear magnetic resonance (STD NMR) epitope mappings with docking analysis, using the α(1 → 3/4)-linked digalactosides and also Galα(1 → 6)Glc (melibiose) as test compounds. The experimental part revealed interaction with the canonical lectin site, and this preferentially via the non-reducing-end galactose moiety. Low-energy conformers appear to be selected without notable distortion, as shown by molecular dynamics simulations. With the α(1 → 4) disaccharide, however, the typical CH-π interaction is significantly diminished, yet binding appears to be partially compensated for by hydrogen bonding. Overall, these findings reveal that the type of α-linkage in digalactosides has an impact on maintaining CH-π interactions and the pattern of hydrogen bonding, explaining preference for the α(1 → 3) linkage. Thus, this lectin is able to accommodate both α- and β-linked galactosides at the same site, with major contacts to the non-reducing-end sugar unit.     

Seasonal dynamics of soil CO<Subscript>2</Subscript> efflux in a conventional tilled wheat field of the Loess Plateau,China

An automated chamber system was employed to measure the soil CO₂ efflux (SCE) in situ for 2 years in a conventional wheat field of the Loess Plateau, China under semi-arid conditions. The annual mean SCE values were 2.44 ± 2.52 μmol m⁻² s⁻¹ in 2006 and 2.37 ± 2.33 μmol m⁻² s⁻¹ in 2007. Distinct seasonality in the SCE was observed, with significant differences occurring among four periods divided by harvesting, tillage and sowing. In the period from tillage to sowing, the mean SCE values were 2.82 and 2.69 times the annual mean values in 2006 and 2007, respectively, and SCE accounted for 39% and 48% of the annual total within 14% and 18% of the days of the years. Although there were significant exponential correlations between the SCE and soil temperature, and significant linear correlations between the SCE and soil moisture for measurements conducted in the periods before tillage and after sowing each year, the SCE from tillage to sowing was significantly beyond the correlation curves. These findings indicated that seasonal variation in the SCE in a conventional field was controlled not only by soil temperature and moisture, but also by tillage practice. The confounding effects of climate and practice on SCE should be considered when developing ways to mitigate soil carbon loss in conventional cropland in semi-arid regions.