Identification of a new gene encoding 5-enolpyruvylshikimate-3-phosphate synthase using genomic library construction strategy

Applying the genomic library construction strategy and colony screening, a new aroA gene encoding 5-enolpyruvylshikimate-3-phosphate synthase has been identified, cloned and overexpressed in Escherichia coli, and the enzyme was purified to homogeneity. Kinetic analysis of the AroA _{P.fluorescens} indicated that the full-length enzyme exhibits 10-fold increased IC50 and an approximately 38-fold increased K _i for glyphosate compared to those of the AroA _E.coli , while retaining high affinity for the substrate phosphoenolpyruvate. Furthermore, we have transformed the new aroA _{P.fluorescens} gene into Arabidopsis thaliana via a floral dip method, and demonstrated that transgenic A. thaliana plants exhibit significant glyphosate resistance when compared with the wild type.     

Novel SNPs in the <Emphasis Type="Italic">PRDM16</Emphasis> gene and their associations with performance traits in chickens

The PR domain containing 16 (PRDM16) is a member of the Prdm family, and is known to regulate cell differentiation. In the present study, DNA pool sequencing methods were employed to screen genetic variations in the chicken PRDM16 gene. The results revealed four novel single nucleotide polymorphisms (SNPs): NC_006108.2: g.92188G>A, XM_417551: c.1161C>T (Ala/Ala, 387aa), c.1233C>T (Ser/Ser, 411aa) and c.1433G>A (Ser/Asn, 478aa). The BglI polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) was used to detect c.1161C>T, while HhaI Forced PCR-RFLP methods were used to detect 1233C>T and c.1433G>A in 964 chickens. The chickens comprised 38 grandparents, 66 F₁ parents and 860 F₂ birds derived from an F₂ resource population of Gushi chickens crossed with Anka broilers. The associations of the polymorphisms in the chicken PRDM16 gene with performance traits were analyzed in the 860 F₂ chickens. The results indicated that the three SNPs were significantly associated with growth, fatness and meat quality traits in the chickens. In particular, the polymorphisms of the missense SNP (c.1433G>A) had positive effects on chicken body weight and body size at different stages. It affected also fatness traits significantly. Comparison of the different genotypes of c.1433G>A showed that the GG genotype favored chicken growth and fatness traits.     

The Ratio of sTfR/Ferritin is Associated with the Expression Level of TfR in Rat Bone Marrow Cells After Endurance Exercise

Currently, it is unclear which index of haematological parameters could be used to most easily monitor iron deficiency during endurance training. To address this question, 16 male Wistar rats were randomly assigned to two groups: a sedentary group (n = 8) and an exercised group (n = 8). Initially, animals in the exercise group started running on a treadmill at a rate of 30 m/min, on a 0% grade, for 1 min/session. Running time was gradually increased by 2 min/day. The training plan was one session per day during the initial 2 weeks and two sessions per day during the third to ninth week. At the end of the 9-week experiment, we analysed the blood of the experimental animals for haemoglobin levels, erythrocyte numbers, haematocrit, serum iron levels, total iron binding capacity, transferrin saturation, serum ferritin levels and soluble transferrin receptor (sTfR) levels, and we calculated the ratio of sTfR/ferritin. Erythrocyte numbers, haemoglobin levels and haematocrit values were decreased after 9 weeks of exercise, but sTfR and sTfR/ferritin values were increased (P < 0.01 or P < 0.05). The training regime significantly increased TfR mRNA levels in the bone marrow cells of the exercised rats compared with the sedentary group (1.8 ± 0.5 vs. 1.1 ± 0.2, P < 0.01). These results revealed a significant correlation between TfR levels in the bone marrow cells and the ratio of sTfR/ferritin (r = 0.517; P < 0.01) and sTfR levels (r = 0.206; P < 0.05) in sedentary and exercised rats. In conclusion, we show that sTfR indices and the ratio of sTfR/ferritin could be useful indicators for monitoring iron deficiency during endurance training.     

Protecting effect of phosphorylation on oxidative damage of D1 protein by down-regulating the production of superoxide anion in photosystem II membranes under high light

The physiological significance of photosystem II (PSII) core protein phosphorylation has been suggested to facilitate the migration of oxidative damaged D1 and D2 proteins, but meanwhile the phosphorylation seems to be associated with the suppression of reactive oxygen species (ROS) production, and it also relates to the degradation of PSII reaction center proteins. To more clearly elucidate the possible protecting effect of the phosphorylation on oxidative damage of D1 protein, the degradation of oxidized D1 protein and the production of superoxide anion in the non-phosphorylated and phosphorylated PSII membranes were comparatively detected using the Western blotting and electron spin resonance spin-trapping technique, respectively. Obviously, all of three ROS components, including superoxide anion, hydrogen peroxide and hydroxyl radical are responsible for the degradation of oxidized D1 protein, and the protection of the D1 protein degradation by phosphorylation is accompanied by the inhibition of superoxide anion production. Furthermore, the inhibiting effect of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), a competitor to Q(B), on superoxide anion production and its protecting effect on D1 protein degradation are even more obvious than those of phosphorylation. Both DCMU effects are independent of whether PSII membranes are phosphorylated or not, which reasonably implies that the herbicide DCMU and D1 protein phosphorylation probably share the same target site in D1 protein of PSII. So, altogether it can be concluded that the phosphorylation of D1 protein reduces the oxidative damage of D1 protein by decreasing the production of superoxide anion in PSII membranes under high light.     

Application of pyrosequencing for Salmonella enterica rapid identification

Application of pyrosequencing of six Salmonella-specific genes as a rapid Salmonella identification method was tested. Primers for hns, hisJ and hilA had non-specific reactions with non-Salmonella strains. Primers for invA, iroB and fimY had specific PCR products and pyrosequences of Salmonella, suggesting that they were suitable for Salmonella rapid identification.     

Real-time analysis of the interaction of a multiple-epitope peptide with antibodies against classical swine fever virus using surface plasmon resonance

The E2 envelope glycoprotein is the major immunodominant protein of classical swine fever virus (CSFV), and can induce neutralizing antibodies and protective immune responses in infected swine. We developed a tandem-repeat multiple-epitope recombinant protein that contains two copies of each of the regions of E2 spanned by residues 693-704, 770-780, and 826-843, coupled by two copies of the region spanned by residues 1446-1460 of the CSFV nonstructural protein NS2-3. The chemically synthesized gene was expressed in Escherichia coli as a fusion with glutathione S-8 (GST), named GST-BT21. After it was purified with Glutathione Sepharose 4B, we used Western blotting to characterize the construct and surface plasmon resonance to analyze its affinity and specific interaction with CSFV-positive serum. Purified GST-BT21 protein displayed excellent immunoreactivity with antiserum against CSFV (Tian et al., 2012), and surface plasmon resonance confirmed the specific affinity between BT21, but not GST, and antibodies in serum from animals infected with CSFV. Surface plasmon resonance is a sensitive and precise method for epitope evaluation, and it can be used to characterize the immunogenicity and functions of recombinant proteins.     

Improvement of bacterial cellulose production by manipulating the metabolic pathways in which ethanol and sodium citrate involved

Nowadays, bacterial cellulose has played more and more important role as new biological material for food industry and medical and industrial products based on its unique properties. However, it is still a difficult task to improve the production of bacterial cellulose, especially a large number of byproducts are produced in the metabolic biosynthesis processes. To improve bacterial cellulose production, ethanol and sodium citrate are added into the medium during the fermentation, and the activities of key enzymes and concentration of extracellular metabolites are measured to assess the changes of the metabolic flux of the hexose monophosphate pathway (HMP), the Embden–Meyerhof–Parnas pathway (EMP), and the tricarboxylic acid cycle (TCA). Our results indicate that ethanol functions as energy source for ATP generation at the early stage of the fermentation in the HMP pathway and the supplementation of ethanol significantly reduces glycerol generation (a major byproduct). While in the EMP pathway, sodium citrate plays a key role, and its supplementation results in the byproducts (mainly acetic acid and pyruvic acid) entering the gluconeogenesis pathway for cellulose synthesis. Furthermore, by adding ethanol and sodium citrate, the main byproduct citric acid in the TCA cycle is also reduced significantly. It is concluded that bacterial cellulose production can be improved by increasing energy metabolism and reducing the formation of metabolic byproducts through the metabolic regulations of the bypasses.     

残锷线蛱蝶线粒体基因组全序列及其系统学意义(英文)

对残锷线蛱蝶(Parathymasulpitia)(鳞翅目:蛱蝶科)线粒体基因组全序列进行了测定。结果表明:残锷线蛱蝶线粒体基因组全序列全长为15268bp,除了在trnS1(AGN)和trnE基因之间有一段121bp长的基因间隔外,其基因的排列顺序及排列方向与大多数已测鳞翅目物种基本一致。在蛋白质编码基因中,除cox1以CGA作为其起始密码子之外,其余12个蛋白质编码基因都以标准的ATN作为起始密码子。此外,除nad4基因以单独的T为终止密码子,其余12个蛋白质编码基因都以TAA结尾。除trnS1(AGN)缺少DHU臂之外,22个tRNA基因都显示典型的三叶草形二级结构。除A+T富集区外的非编码序列中,线粒体基因组共含有11个基因间隔区。其中,最长的一个121bp的基因间隔区位于trnS1(AGN)和trnE之间,其A+T含量高达100%。另外,和其他鳞翅目物种一样,在其A+T富集区的3’端有一段长达18bp的poly-T结构。A+T富集区内部没有明显的小卫星样多拷贝重复序列,而含有一些微卫星样的重复结构。本研究基于13种蛋白编码基因序列的组合数据,用最大似然法和贝叶斯法对蛱蝶科几个主要亚科间共9个代表物种间的系统发生关系进行了分析。结果表明,本研究的结果与前人的分子系统学研究结论基本吻合(其中,线蛱蝶亚科和釉蛱蝶亚科互为姐妹群),而与形态学的研究结论不一致。     

Endoglin mediates fibronectin/α5β1 integrin and TGF-β pathway crosstalk in endothelial cells

Both the transforming growth factor β (TGF-β) and integrin signalling pathways have well-established roles in angiogenesis. However, how these pathways integrate to regulate angiogenesis is unknown. Here, we show that the extracellular matrix component, fibronectin, and its cellular receptor, α5β1 integrin, specifically increase TGF-β1- and BMP-9-induced Smad1/5/8 phosphorylation via the TGF-β superfamily receptors endoglin and activin-like kinase-1 (ALK1). Fibronectin and α5β1 integrin increase Smad1/5/8 signalling by promoting endoglin/ALK1 cell surface complex formation. In a reciprocal manner, TGF-β1 activates α5β1 integrin and downstream signalling to focal adhesion kinase (FAK) in an endoglin-dependent manner. α5β1 integrin and endoglin form a complex on the cell surface and co-internalize, with their internalization regulating α5β1 integrin activation and signalling. Functionally, endoglin-mediated fibronectin/α5β1 integrin and TGF-β pathway crosstalk alter the responses of endothelial cells to TGF-β1, switching TGF-β1 from a promoter to a suppressor of migration, inhibiting TGF-β1-mediated apoptosis to promote capillary stability, and partially mediating developmental angiogenesis in vivo. These studies provide a novel mechanism for the regulation of TGF-β superfamily signalling and endothelial function through crosstalk with integrin signalling pathways.