Detection and Quantification of Fusarium oxysporum f. sp. niveum Race 1 in Plants and Soil by Real-time PCR

Fusarium wilt caused by Fusarium oxysporum f. sp. niveum (Fon) is the most serious soil-borne disease in the world and has become the main limiting factor of watermelon production. Reliable and quick detection and quantification of Fon are essential in the early stages of infection for control of watermelon Fusarium wilt. Traditional detection and identification tests are laborious and cannot efficiently quantify Fon isolates. In this work, a real-time polymerase chain reaction (PCR) assay has been described to accurately identify and quantify Fon in watermelon plants and soil. The FONRT-18 specific primer set which was designed based on identified specific sequence amplified a specific 172 bp band from Fon and no amplification from the other formae speciales of Fusarium oxysporum tested. The detection limits with primers were 1.26 pg/μl genomic DNA of Fon, 0.2 pg/ng total plant DNA in inoculated plant, and 50 conidia/g soil. The PCR assay could also evaluate the relationships between the disease index and Fon DNA quantity in watermelon plants and soil. The assay was further used to estimate the Fon content in soil after disinfection with CaCN₂. The real-time PCR method is rapid, accurate and reliable for monitoring and quantification analysis of Fon in watermelon plants and soil. It can be applied to the study of disease diagnosis, plant-pathogen interactions, and effective management.     

The affinity of human RANK binding to its ligand RANKL

Receptor activator of nuclear factor-kappa B (RANK) and its ligand, RANKL play critical roles in bone re-modeling, immune function, vascular disease and mammary gland development. To study the interaction of RANK and RANKL, we have expressed both extracellular domain of RANK and ectodomain of RANKL using Escherichia coli expression system. RANK was expressed as an inclusion body first which properly refolded later, while RANKL was initially produced as a GST fusion protein, after which the GST was removed by enzyme digestion. Soluble RANK existed as a monomer while RANKL was seen as a trimer in solution, demonstrated by gel filtration chromatography and cross-linking experiment. The recombinant RANK and RANKL could bind to each other and the binding affinity of RANKL for RANK was measured with surface plasmon resonance technology and K_D value is about 1.09 × 10⁻¹⁰ M.     

Phenotypic responses of hatchlings to constant versus fluctuating incubation temperatures in the multi-banded krait, Bungarus multicintus (Elapidae)

Most studies on egg incubation in reptiles have relied on constant temperature incubation in the laboratory rather than on simulations of thermal regimes in natural nests. The thermal effects on embryos in constant-temperature studies often do not realistically reflect what occurs in nature. Recent studies have increasingly recognized the importance of simulating natural nest temperatures rather than applying constant-temperature regimes. We incubated Bungarus multicintus eggs under three constant and one fluctuating-temperature regimes to evaluate the effects of constant versus fluctuating incubation temperatures on hatching success and hatchling phenotypes. Hatching success did not differ among the four treatments, and incubation temperature did not affect the sexual phenotype of hatchlings. Incubation length decreased as incubation temperature increased, but eggs incubated at fluctuating temperatures did not differ from eggs incubated at constant temperatures with approximately the same mean in incubation length. Of the hatchling phenotypes examined, residual yolk, fat bodies and locomotor performance were more likely affected by incubation temperature. The maximal locomotor speed was fastest in the fluctuating-temperature and 30 degrees C treatments and slowest in the 24 degrees C treatment, with the 27 degrees C treatment in between. The maximal locomotor length was longest in the fluctuating-temperature treatment and shortest in the 24 degrees C and 27 degrees C treatments, with the 30 degrees C treatment in between. Our results show that fluctuating incubation temperatures do not influence hatching success and hatchling size and morphology any differently than constant temperatures with approximately the same mean, but have a positive effect on locomotor performance of hatchlings.     

Crystal structure of type VI effector Tse1 from Pseudomonas aeruginosa

The type VI secretion systems (T6SS) have emerging roles in interspecies competition. In order to have an advantage in defense against other organisms, this system in Pseudomonas aeruginosa delivers a peptidoglycan amidase (Tse1) to the periplasmic space of a competitor. An immune protein (Tsi1) is also produced by the bacterium to protect itself from damage caused by Tse1. Tsi1 directly interacts with Tse1. We report that the crystal structure of Tse1 displays a common CHAP protein fold. Strikingly, our structures showed that the third residue in the catalytic triad may be novel as this residue type has not been observed previously.     

鱼类多样性监测的理论方法及中国内陆 水体鱼类多样性监测

近年来, 生物多样性监测网络的建设得到广泛重视, 全球、地区或国家生物多样性观测网不断组建。生物多样性观测的理论框架得到发展, 提出了生物多样性核心监测指标(Essential Biodiversity Variables, EBV)。鱼类多样性监测的理论框架包含于生物多样性核心监测指标之内, 在遗传、物种、生态系统等多层次进行。基于鱼类监测提出的生物完整性指数(index of biotic integrity, IBI)强调不同物种的生态功能, 可以综合反映群落结构和功能的变化, 得到广泛应用。鱼类多样性的监测方法是传统网具和现代水声学等方法的结合。监测结果的分析可以进行简单的指数比较, 也可以进行长期的趋势分析, 寻找关键节点, 探讨宏观生态格局的变化。中国内陆水体鱼类多样性监测网隶属于中国生物多样性监测与研究网络, 拟选取长江、黄河、黑龙江、珠江、澜沧江、怒江、塔里木河及青海湖8大流域, 对25个重要区域和24个重点物种(类群)进行监测, 从重要区域鱼类群落结构、重点物种(类群)种群动态和个体生物学特征、遗传多样性、早期资源等不同层次, 全面监测我国内陆水体鱼类生物多样性状况。     

成年大鼠海马CA1区锥体细胞KATP通道的特性

为了解成年大鼠海马CA1区锥体细胞KATP通道的特性,实验采用膜片钳技术的内面向外式记录法,在急性分离的CA1区锥体神经元上,研究了可被胞浆侧ATP所抑制的钾离子单通道的特性,当细胞膜内外两侧的K^ 浓度均为140mmol/L时,通道的电导为63pS,翻转电位为1．71mV,通道呈弱向内向整流性,在负钳制电位时,通道开放时常被短时的关闭所打断,而在正钳制电位时,这种短时程的关闭状态明显少于负钳制电位时,但通道开放概率未见明显的电压依赖性,ATP对通道活动的抑制作用呈浓度依赖性,抑制通道活动50％的ATP浓度为0．1mmol/L.KATP通道的特异性阻断剂tolbutamide(甲糖宁,1mmol/L)可完全阻断通道的活动,而KATP通道开放剂diazoxide(二氮嗪,1mmol/L）则不增强通道的活动。     

Purification and characterization of a novel chlorpyrifos hydrolase from Cladosporium cladosporioides Hu-01

Chlorpyrifos is of great environmental concern due to its widespread use in the past several decades and its potential toxic effects on human health. Thus, the degradation study of chlorpyrifos has become increasing important in recent years. A fungus capable of using chlorpyrifos as the sole carbon source was isolated from organophosphate-contaminated soil and characterized as Cladosporium cladosporioides Hu-01 (collection number: CCTCC M 20711). A novel chlorpyrifos hydrolase from cell extract was purified 35.6-fold to apparent homogeneity with 38.5% overall recovery by ammoniumsulfate precipitation, gel filtration chromatography and anion-exchange chromatography. It is a monomeric structure with a molecular mass of 38.3 kDa. The pI value was estimated to be 5.2. The optimal pH and temperature of the purified enzyme were 6.5 and 40°C, respectively. No cofactors were required for the chlorpyrifos-hydrolysis activity. The enzyme was strongly inhibited by Hg2?, Fe3?, DTT, β-mercaptoethanol and SDS, whereas slight inhibitory effects (5-10% inhibition) were observed in the presence of Mn2?, Zn2?, Cu2?, Mg2?, and EDTA. The purified enzyme hydrolyzed various organophosphorus insecticides with P-O and P-S bond. Chlorpyrifos was the preferred substrate. The Km and Vmax values of the enzyme for chlorpyrifos were 6.7974 μM and 2.6473 μmol·min?1, respectively. Both NH2-terminal sequencing and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometer (MALDI-TOF-MS) identified an amino acid sequence MEPDGELSALTQGANS, which shared no similarity with any reported organophosphate-hydrolyzing enzymes. These results suggested that the purified enzyme was a novel hydrolase and might conceivably be developed to fulfill the practical requirements to enable its use in situ for detoxification of chlorpyrifos. Finally, this is the first described chlorpyrifos hydrolase from fungus.     

Removal of selectable marker gene from fibroblast cells in transgenic cloned cattle by transient expression of Cre recombinase and subsequent effects on recloned embryo development

Introduction of selectable marker genes to transgenic animals could create an inconvenience to further research and may exaggerate public concerns regarding biological safety. The objective of the current study was to excise loxP flanked neo^R in transgenic cloned cattle by transient expression of Cre recombinase. Green fluorescent protein gene (GFP) was incorporated to monitor Cre expression; therefore, Cre-expressed cells could be selected indirectly by fluorescence-activated cell sorting (FACS). The neo^R was removed and Cre expressed transiently in GFP-positive colonies; excision of neo^R was confirmed by single-blastocyst PCR in recloned blastocysts, with neo^R-free fibroblast cells as donors. There was no difference (P > 0.05) in rates of cleavage (76.0% vs. 68.8%) or blastocyst formation (56.6% vs. 52.9%) between recloned embryos with neo^R-free or neo^R-included donors. The differential staining of recloned blastocysts were similar (P >0.05) in terms of total cell number (124 vs. 122) and the ratio of ICM (Inner Cell Mass) to the total cell number (38.1% vs. 38.2%). Furthermore, pregnancy and calving rates were not different (P > 0.05) from those of the control. In conclusion, we successfully excised neo^R from transgenic cloned cattle; the manipulation did not affect the developmental competence of recloned preimplantation embryos. This approach should benefit bioreactor and transgenic research in livestock.     

Amplification activation loop between caspase-8 and -9 dominates artemisinin-induced apoptosis of ASTC-a-1 cells

Although caspases have been demonstrated to be involved in artemisinin (ARTE)-induced apoptosis, their exact functions are not well understood. The aim of this report is to explore the roles of caspase-8, -9 and -3 during ARTE-induced apoptosis in human lung adenocarcinoma (ASTC-a-1) cells. ARTE treatment induces a rapid generation of reactive oxygen species (ROS), and ROS-dependent apoptosis as well as the activation of caspase-8, -9 and -3 via time- and dose-dependent fashion. Of upmost importance, inhibition of caspase-8 or -9, but not caspase-3, almost completely blocks the ARTE-induced not only activation of the caspase-8, -9 and -3 but also apoptosis. In addition, the apoptotic process triggered by ARTE does not involve the Bid cleavage, tBid translocation, significant loss of mitochondrial membrane potential and cytochrome c release from mitochondria. Moreover, silencing Bax/Bak does not prevent the ATRE-induced cell death as well as the activation of caspase-8, -9 and -3. Collectively, our data firstly demonstrate that ARTE triggers a ROS-mediated positive feedback amplification activation loop between caspase-8 and -9 independent of mitochondria, which dominantly mediated the ARTE-induced apoptosis via a caspase-3-independent apoptotic pathway in ASTC-a-1 cells. Our findings imply a potential to develop new derivatives from artemisinin to effectively initiate the amplification activation loop of caspases.