Selection and proliferation of rapid growing cell lines from embryo derived cell cultures of yew tree (Taxus cuspidata Sieb. et Zucc)

Calli were induced from 300,000 embryos isolated from immature to mature stage of seeds collected on late September from 14 elite trees. When the embryos were cultured onto plastic Petri-dish containing 20 mL of modified B5 basal medium supplemented with 3% (w/v) sucrose, 500 mg/L casein hydrolysate, 250 mg/L myo-inositol, 0.5% (w/v) polyvinyl polypyrrolidon (PVPP), 2×MS vitamins, 0.5 mg/L gibberellic acid, and 10 mg/L 2,4-D after 2 weeks of culture, yellowish-white calli were immediately formed on the surfaces of embryos, and subcultured for 4 weeks in same culture medium. Because most of calli maintained for more than 3 months were revealed differences in their colors, surface texture, and growth rate, visual selection was made for first round screening. When the size of visually selected calli larger than 19 mm in their diameter were inoculated, persistent proliferation was observed. Among the plating methods tested for the selection of rapid growing cell lines at single cell and/or small cell aggregate level, 2-layer spread plating revealed as the best for single cell cloning. To enhance cell growth and maintain high rate of viability for long-term culture of yew cells in bioreactor, final cell volume less than 50% in SCV seemed to be the best. Time course study revealed that 30% of inoculum density was suitable for fed batch culture. Among the tested conditional media, the rate of 1∶2 (old medium: fresh medium) was recorded at the best for cell growth.     

Cascade control of Escherichia coli glutamine synthetase. Purification and properties of PII protein and nucleotide sequence of its structural gene

A procedure was developed to purify large quantities of PII protein from an Escherichia coli strain which contains a multicopy plasmid harboring the structural gene of PII (the glnB gene). Ultraviolet spectra of uridylylated and unuridylylated PII were obtained using the purified PII and empirical formulas to calculate the concentration of protein and the average number of uridylylated subunits per molecule were derived. A continuous fluorometric assay for the measurement of uridylylated PII (PIID) and adenylyltransferase (ATase) was also established. Rate measurements at various concentrations of PIID and at a fixed concentration of ATase showed that a tetrameric PIID molecule interacts with only one ATase molecule at a time. The complete nucleotide sequence of the glnB gene was determined and parts of the deduced amino acid sequence were confirmed by the results of amino acid sequence analysis of peptides. The PII subunit consists of 103 amino acids (Mr = 11,580). Two tyrosines reside at positions 46 and 51, where Tyr51 is the site of uridylylation. Nucleotide sequence analysis of the upstream region showed no obvious sites for the binding of RNA polymerase, indicating that the glnB gene is a part of an as yet unidentified operon.     

AvrRps4 effector family processing and recognition in lettuce

During pathogenesis, effector proteins are secreted from the pathogen to the host plant to provide virulence activity for invasion of the host. However, once the host plant recognizes one of the delivered effectors, effector‐triggered immunity activates a robust immune and hypersensitive response (HR). In planta, the effector AvrRps4 is processed into the N‐terminus (AvrRps4^N) and the C‐terminus (AvrRps4^C). AvrRps4^C is sufficient to trigger HR in turnip and activate AtRRS1/AtRPS4‐mediated immunity in Arabidopsis; on the other hand, AvrRps4^N induces HR in lettuce. Furthermore, AvrRps4^N‐mediated HR requires a conserved arginine at position 112 (R112), which is also important for full‐length AvrRps4 (AvrRps4^F) processing. Here, we show that effector processing and effector recognition in lettuce are uncoupled for the AvrRps4 family. In addition, we compared effector recognition by lettuce of AvrRps4 and its homologues, HopK1 and XopO. Interestingly, unlike for AvrRps4 and HopK1, mutation of the conserved R111 in XopO by itself was insufficient to abolish recognition. The combination of amino acid substitutions arginine 111 to leucine with glutamate 114 to lysine abolished the XopO‐mediated HR, suggesting that AvrRps4 family members have distinct structural requirements for perception by lettuce. Together, our results provide an insight into the processing and recognition of AvrRps4 and its homologues.     

nAdder: A scale-space approach for the 3D analysis of neuronal traces

Tridimensional microscopy and algorithms for automated segmentation and tracing are revolutionizing neuroscience through the generation of growing libraries of neuron reconstructions. Innovative computational methods are needed to analyze these neuronal traces. In particular, means to characterize the geometric properties of traced neurites along their trajectory have been lacking. Here, we propose a local tridimensional (3D) scale metric derived from differential geometry, measuring for each point of a curve the characteristic length where it is fully 3D as opposed to being embedded in a 2D plane or 1D line. The larger this metric is and the more complex the local 3D loops and turns of the curve are. Available through the GeNePy3D open-source Python quantitative geometry library (https://genepy3d.gitlab.io), this approach termed nAdder offers new means of describing and comparing axonal and dendritic arbors. We validate this metric on simulated and real traces. By reanalysing a published zebrafish larva whole brain dataset, we show its ability to characterize different population of commissural axons, distinguish afferent connections to a target region and differentiate portions of axons and dendrites according to their behavior, shedding new light on the stereotypical nature of neurites’ local geometry.     

半干旱区沙质退化草地造林对土壤质量的影响

采用野外调查与室内培养相结合的方法,研究了我国北方半干旱区科尔沁沙地退化草地营造樟子松人工林32年后0～10 cm表层土壤理化性状、土壤碳氮矿化量、土壤微生物量以及土壤酶活性等的变化. 结果表明32年生樟子松人工林土壤有机碳、全氮和全磷等养分含量分别下降了21%、42%和45%;5月和11月樟子松人工林土壤NH4 -N显著高于草地(P=0.001;P=0.019),而5、8和11月草地土壤NO3--N含量显著高于樟子松人工林(P＜0.001;P=0.048;P=0.031);5、8和11月樟子松人工林土壤有机碳日矿化释放的CO2-C量均大于草地,而二者土壤氮矿化率差异不显著(P＞0.05);5和8月樟子松人工林土壤微生物量碳含量与草地相比差异不显著,11月则显著高于草地;土壤养分和水分含量是影响土壤微生物量碳含量的重要因素;与草地相比,樟子松人工林土壤脲酶和蔗糖酶活性降低,而土壤过氧化氢酶活性升高. 上述结果说明半干旱区沙质退化草地营造樟子松人工林32年后土壤质量出现一定程度的下降;由于植被的改变,樟子松人工林土壤理化性状和生物学性状等表现出与草地不同的季节动态特征.造林作为我国北方半干旱区沙地退化生态系统的一种恢复手段具有一定的局限性.     

温度对中型指环虫产卵和孵化的影响

指环虫病是严重影响鱼类养殖的寄生虫病.为了有效控制指环虫病,实验研究了寄生在金鱼(Carassius auratus)鳃部的中型指环虫(Dactylogyrus intermedius)卵、纤毛幼虫的形态,以及在离体条件下温度对其产卵和孵化的影响.成熟的中型指环虫虫卵大部分为梨形,长30 μm左右,后端有一个卵柄.纤毛幼虫呈圆筒状,两端稍尖,眼点两对;后吸盘具有若干对小锚钩;在前部、中部和尾部分别具有一圈纤毛.实验研究了4℃、10℃、22℃、30℃和35℃条件下中型指环虫的产卵和孵化情况,在4℃条件下,中型指环虫基本不产卵也不孵化;在其他4个温度条件下,产卵量随着温度的升高而增加,其平均产卵量分别为3.30、4.10、4.13和4.24枚/虫.统计结果显示:在35℃条件下的产卵量明显高于10℃(P＜0.05),其他温度条件下的平均产卵量没有显著性差异.中型指环虫的产卵速率随着温度的升高而加快,产卵维持的时间分别为4d、23h、15h和llh.孵化率在22℃时最高,为72.7％,在30℃和35℃的孵化率为50％左右,卡方检验显示:4种温度下的孵化率之间没有显著性差异p＞0.05);随着温度的升高,孵化速率逐渐加快,而孵化时间和纤毛幼虫的存活时间则缩短,平均孵化时间分别为24d、3d、42h和26h,纤毛幼虫的最长存活时间分别为4d、3d、56h和34h.结果显示,当水温为22℃时,中型指环虫的产卵数量和纤毛幼虫的存活时间都比较高,且孵化率最高,表明该温度条件较适合中型指环虫的种群增长.     

One-step knockin for inducible expression in mouse embryonic stem cells

Transgenesis enables the elucidation of gene function; however, constant transgene expression is not always desired. The tetracycline responsive system was devised to turn on and off transgene expression at will. It has two components: a doxycycline (dox)-controlled transactivator (TA) and an inducible expression cassette. Integration of these transgenes requires two transfection steps usually accomplished by sequential random integration. Unfortunately, random integration can be problematic due to chromatin position effects, integration of variable transgene units, and mutation at the integration site. Therefore, targeted transgenesis and knockin were developed to target the TA and the inducible expression cassette to a specific location, but these approaches can be costly in time, labor, and money. Here, we describe a one-step Cre-mediated knockin system in mouse embryonic stem cells that positions the TA and inducible expression cassette to a single location. Using this system, we show dox-dependent regulation of eGFP at the DNA topoisomerase 3β promoter. Because Cre-mediated recombination is used in lieu of gene targeting, this system is fast and efficient.     

食品微生物本科毕业论文实验的探索与实践——以干酪乳杆菌异源表达胆盐水解酶为例

毕业论文是本科教育教学过程中不可替代的重要环节之一,是检验学生综合素质的重要手段。以干酪乳杆菌异源表达胆盐水解酶为例,从论文选题、实验准备、实验研究和论文撰写等方面介绍上海理工大学食品微生物本科毕业论文实验的有关探索。学生通过综合运用所学理论知识和实验技能,能够顺利完成毕业论文实验,构建出具有工业应用潜力的耐胆盐干酪乳杆菌基因工程菌,达到本科毕业论文实践教学的目的。     

Cyclic guanosine-3',5'-monophosphate and biopteridine biosynthesis in Nocardia sp

Nocardia sp. strain NRRL 5646 contains a nitric oxide synthase (NOS) enzyme system capable of generating nitric oxide (NO) from arginine and arginine-containing peptides. To explain possible roles of the NOS system in this bacterium, guanylate cyclase (GC) and tetrahydrobiopterin (H(4)B) biosynthetic enzymes were identified in cell extracts and in culture media. Cell extracts contained GC activity, as measured by the conversion of GTP to cyclic guanosine-3',5'-monophosphate (cGMP) at 9.56 pmol of cGMP h(-1) mg of protein(-1). Concentrations of extracellular cGMP in culture media were significantly increased, from average control levels of 45 pmol cGMP liter(-1) to a maximum of 315 pmol liter(-1), in response to additions of GTP, L-arginine, H(4)B, and sodium nitroprusside to growing Nocardia cultures. On the other hand, the NOS inhibitor N(G)-nitro-L-arginine and the GC inhibitor 1H-[1,2, 4]oxadiazole[4,3-a]quinoxalin-1-one both dramatically decreased extracellular cGMP levels. Activities for GTP-cyclohydrase-1, 6-pyruvoyltetrahydropterin synthase and sepiapterin reductase, enzymes essential for H(4)B biosynthesis, were present in Nocardia culture extracts at 77.5 pmol of neopterin and 45.8 pmol of biopterin h(-1) mg of protein(-1), respectively. In Nocardia spp., as in mammals, GTP is a key intermediate in H(4)B biosynthesis, and GTP is converted to cGMP by a GC enzyme system that is activated by NO.