A novel and highly efficient production system for recombinant adeno-associated virus vector

Recombinant adeno-associated virus(rAAV) has proven to be a promising gene delivery vector for human gene therapy. However, its application has been limited by difficulty in obtaining enough quantities of high-titer vector stocks. In this paper, a novel and highly efficient production system for rAAV is described. A recombinant herpes simplex virus type 1(rHSV-1) designated HSV1-rc/△UL2, which expressed adeno-associated virus type2(AAV-2) Rep and Cap proteins, was constructed previously. The data confirmed that its functions were to support rAAV replication and packaging, and the generated rAAV was infectious. Meanwhile, an rAAV proviral cell line designated BHK/SG2, which carried the green fluorescent protein(GFP) gene expression cassette, was established by transfecting BHK-21 cells with rAAV vector plasmid pSNAV-2-GFP. Infecting BHK/SG2 with HSV1-rc/△UL2 at an MOI of 0.1 resulted in the optimal yields of rAAV, reaching 250 transducing unit(TU) or 4.28×104 particles per cell. Therefore, compared with the conventional transfection method, the yield of rAAV using this "one proviral cell line, one helper virus" strategy was increased by two orders of magnitude. Large-scale production of rAAV can be easily achieved using this strategy and might meet the demands for clinical trials of rAAV-mediated gene therapy.     

Possible role of ionic gradients in the apical growth of <Emphasis Type="Italic">Neurospora crassa</Emphasis>

The effects of the Ca²⁺/H⁺ exchanger A23187 and the K⁺/H⁺ exchanger nigericin on the growth of Neurospora crassa were analyzed. Both ionophores had the same effects on the fungus. They both inhibited growth in liquid media, apical extension being more affected than protein synthesis. A sudden challenge to either ionophore on solid media rapidly stopped hyphal extension. Additionally, both ionophores induced profuse mycelium branching and upward hyphal growth. Hyphae growing on nigericin-containing media also burst at the apex. Both ionophores caused a rapid inhibition in the apically-occurring synthesis of structural wall polysaccharides, but they did not affect mitochondrial energy conservation. With the use of DiBAC, a membrane-potential sensitive fluorophore, it was excluded that their effects were due to depletion of the plasma membrane potential. Considering that both ionophores exchange H⁺ for different metallic ions, we concluded that their effect was due to dissipation of a proton gradient, which is directly or indirectly involved in the apical growth of the fungus. This revised version was published online in June 2006 with corrections to the Cover Date.     

Gene expression profile in immunologically injured liver cell of mice

To study the gene expression profiles between immunologically injured liver cell and normal liver cell of mice and to screen on a large scale the differentially expressed genes associated with the formation of liver injury, the experimental mice were randomly divided into the normal group for controlling and the immunologically liver-injured group induced by BCG and LPS. The liver mRNA of the two groups were extracted respectively and reversely-transcribed to cDNA with the incorporation of different fluorescence (Cy3, Cy5) labeled dUTP as the hybridization probes. The mixed probes were hybridized to the cDNA microarray chips. The fluorescent signal results were acquired by scanner ScanArray 4000 and analyzed with software GenePix Pro 3.0. Among the 14112 target genes, 293 genes were found to be significantly differentially expressed, in which 188 genes were up-regulated and 105 genes were down-regulated. Based on the analysis of biological functions of those differentially expressed genes, it was indicated that the occurrence and development of mouse liver damage induced by BCG and LPS were highly correlated with the processes of immune reactions, cell synthesis, metabolism, apoptosis and transportation in liver cell, which might be quite important for elucidating the regulatory network of gene expression associated with the liver damage, also important for finally discovering the pathogenic mechanisms of immunological liver damage.     

Associations between primates and other mammals in a central Amazonian forest landscape

Little information exists on mixed-species groups between primates and other mammals in Neotropical forests. In this paper, we describe three such associations observed during an extensive large-vertebrate survey in central Amazonia, Brazil. Mixed-species groups between a primate species and another mammal were observed on seven occasions between squirrel monkeys (Saimiri cf. ustus) and either South American coatis (Nasua nasua) or tayras (Eira barbara) and between brown capuchins (Cebus apella) and coatis. All associations were restricted to floodplain forest during its dry stage. We suggest that the associations involving the coatis are connected to foraging and vigilance but may be induced by a common alternative food resource at a time of food shortage.     

通过染色体外同源重组建立乳腺中表达外源基因的转基因小鼠

为研究染色体外重组方法在创建转基因动物中的应用,选取牛asl酪蛋白基因的5′及3′侧翼区和氯霉素乙酰化酶编码区,构建了两个具有3 kb相互重叠的融合基因.将这两个DNA片段末端脱磷酸后,以摩尔比1:1的比例混合,通过显微注射导入小鼠受精原核,最后获得了11个品系的转基因小鼠.对其中10个品系的小鼠的整合分析表明,所注射的两个DNA片段均发生了染色体外同源重组,而且除了 1个品系的小鼠丢失了大约1kb的序列外,其余品系小鼠的重组产物与预想的结构符合.在当代和后代的转基因小鼠乳汁中均可测到氯霉素乙酰化酶的活性.这表明融合基因在转基因小鼠乳腺中得到表达和分泌,也说明显微共注射两个相互重叠的基因片段是建立转基因动物的一个可行途径.     

Observation of brown-throated three-toed sloths: mating behaviour and the simultaneous nurturing of two young

Bradypus variegatus is a member of the Order Pilosa, Family Bradypodidae, and is distributed in many subtropical and tropical countries in South and Central America. However, studies on this species in the wild are relatively limited and many aspects of its reproductive behaviour are unknown or unclear. The current report presents new observations of the reproductive behaviour of B. variegatus in its natural environment. These include details of both a male–female copulation and the simultaneous nurturing of two young sloths.     

Influences of calcium deficiency and cerium on the conversion efficiency of light energy of spinach

Chloroplast absorbs light energy and transforms it into electron energy, and then converts it into active chemical energy and stable chemical energy. In the present paper, we investigated the effects of Ce(3+), which has the most significant catalytic effects and similar characteristics with Ca(2+), on light energy conversion of spinach chloroplasts under Ca(2+)-deficient stress. The results illuminated that the Hill reaction activity, electron flow both photosystems and photophosphorylation rate of spinach chloroplasts reduced significantly under Ca(2+)-deficient condition, and activities of Mg(2+)-ATPase and Ca(2+)-ATPase on the thylakoid membrane were severely inhibited. Meanwhile, the activity of Rubisco, which is the key enzyme of photosynthetic carbon assimilation, was also prohibited. However, Ce(3+) decreased the inhibition of calcium deprivation the electron transport rate, the oxygen evolution rate, the cyclic and noncyclic photophosphorylation, the activities of Mg(2+)-ATPase, Ca(2+)-ATPase and Rubisco of spinach chloroplasts. All above implied that Ca(2+)-depletion could disturb light energy conversion of chloroplasts strongly, which could be reversed by Ce(3+).     

Effect of <Emphasis Type="Italic">poxB</Emphasis> gene knockout on metabolism in <Emphasis Type="Italic">Escherichia coli</Emphasis> based on growth characteristics and enzyme activities

The effect of poxB gene knockout on metabolism in Escherichia coli was investigated in the present paper based on the growth characteristics and the activities of the enzymes involved in the central metabolic pathways. The absence of pyruvate oxidase reduced the glucose uptake rate and cell growth rate, and increased O₂ consumption and CO₂ evolution. The enzyme assay results showed that although glucokinase activity increased, the flux through glycolysis was reduced due to the down-regulation of the other glycolytic enzymes such as 6-phosphofructosekinase and fructose bisphosphate aldolase in the poxB mutant. TCA cycle enzymes such as citrate synthase and malate dehydrogenase were repressed in the poxB mutant when the cells were cultivated in LB medium. The pyruvate oxidase mutation also resulted in the activation of glucose-6-phosphate dehydrogenase and acetyl-CoA synthetase. All these results suggest that pyruvate oxidase is not only a stationary-phase enzyme as previously known, and that the removal of the poxB gene affects the central metabolism at the enzyme level in E. coli.     

A trypsin inhibitor from <Emphasis Type="Italic">Cassia obtusifolia</Emphasis> seeds: isolation,characterization and activity against <Emphasis Type="Italic">Pieris rapae</Emphasis>

A trypsin inhibitor was isolated from Cassia obtusifolia by ammonium sulfate precipitation, Sepharose 4B-trypsin affinity and Sephadex G-75 chromatography. The inhibitor consisted of a single polypeptide chain with a molecular mass of 19, 812.55 Da. It was stable from pH 2 to 12 for 24 h, whereas it was unstable either above 70°C for 10 min or under reduced conditions. The inhibitor, which inhibited trypsin activity with an apparent Ki of 0.3 μM, had one reactive site involving a lysine residue. The native inhibitor was resistant to pepsin digestion, whereas the heated inhibitor produced 40% degree of susceptibility. The disulfide linkage and lysine residue were important in maintaining its conformation. Partial amino acid sequence of the purified protein showed a high degree of homology with various members of the Kunitz inhibitor family. Moreover, the inhibitor showed significant inhibitory activity against trypsin-like proteases present in the larval midgut on Pieris rapae and could suppress the growth of larvae.     

Foot mucus is a good source for non-destructive genetic sampling in Polyplacophora

A non-destructive method for collecting samples for DNA analysis from the mucus of molluscs was successfully adapted for use with the genus Ischnochiton. DNA was extracted using a Chelex-based method and the COI subunit of the mtDNA was amplified and sequenced. Sequences from the mucus were crosschecked against sequences from the foot tissue of the same animal and were found to be identical. This method provides a non-destructive way of carrying out larger studies of the genetics of rare organisms and may be of general use for genetic-based field studies of molluscs.