Purification, Refolding of Hybrid hIFNγ-Kringle 5 Expressed in Escherichia coli

The DNA sequence coding for plasminogen kringle 5 (pK5), an inhibitor of angiogenesis, was fused with that coding for interferon gamma and over-produced in the form of inactive inclusion bodies in E. coli. The amount of fusion protein was about 40% of total protein produced. The fusion protein contained in the inclusion bodies was solubilized in 8 m urea and purified by anion-exchange chromatography. We employed the orthogonal experimental design L₁₆(4⁵) (5 factors, 4 levels, 16 experiments) procedure for researching the influence of denaturant, aggregation suppressor l-arginine, NaCl, pH, and glycine on the refolding procedure. Our results suggest that the presence of appropriate l-arginine, NaCl, and denaturant in the refolding buffer inhibits the aggregation of the fusion protein and increases the yield of renatured protein with biological activity. The refolded fusion protein, γIFN/pk5, has in vitro anti-endothelial cell proliferation activity. Received: 24 July 2000 / Accepted: 21 September 2000     

Isolation of GUS marker lines for genes expressed in Arabidopsis endosperm,embryo and maternal tissues

In order to identify marker lines expressing GUS in various endosperm compartments and at different developmental stages, a collection of Arabidopsis thaliana (L.) Heynh. promoter trap lines were screened. The screen identified 16 lines displaying GUS-reporter gene expression in the endosperm, embryo and other seed organs. The distinctive patterns of GUS expression in these lines provide molecular markers for most cell compartments in the endosperm of Arabidopsis seeds at all developmental stages, and represent a valuable research tool for characterizing present and future Arabidopsis seed mutants. GUS expression patterns of these 16 lines are presented here. One line showed chalazal endosperm-specific GUS activity at the heart stage of embryo development. In six lines embryo-specific GUS activity was detected. Six lines exhibited GUS activity predominantly in the endosperm and embryo while two lines showed strong GUS activity in all seed organs. In one line GUS activity was detected in integuments and syncytial endosperm, while the GUS activity at the cotyledonary stage of the embryo was seed coat-specific. In addition, two funiculus markers and two silique markers expressed in the abscission zone and the guard cells are also presented.     

鼎湖山南亚热带季风常绿阔叶林凋落物的养分动态

本文探讨鼎湖山季风常绿阔叶林调落物养分特征,分析了其动态、季节间与种间变异、构成特征等。结果表明,该群落各种群含氮量的季节变化较小,各个种的变异系数多在15％以下,但磷、钾和钠的变异较大。无论在群落还是在种的水平上,各种营养成份间均没有显著的相关性。除磷外,不同种问养分含量有显著差异,季节间则除镁外无显著差异,磷在红壤地区土壤中难于利用的主要原因可能是不同植物在磷的利用上趋同．全年通过凋落物回归土壤的养分量为氮99．88kghm-2,磷15．03kghm-2．钾27．01kghm-2,钙37．11kghm-2;镁12．19kghm-2。在该群落中,椎栗是对落叶养分贡献最大的种,荷木其次。8月是当年凋落物养分量最高的月份,主要是由于台风作用产生大量的枝叶折落,12月则是最低月份,两者在构成上的主要区别是8月份枝条所持养分量极大,而12月份枝条极少。全年凋落物养分出现两次高峰,4月和8月,而以较寒冷的1、2、11、12月份最低。     

棉花miR397 LAC4模块调控棉铃虫抗性研究

miRNA(microRNA)通过调控其靶标基因在植物的生长、发育和抗逆过程中扮演着重要的角色。该研究采用分子生物学和生物化学等方法,探讨棉花miR397 LAC4参与植株木质素生物合成和对棉铃虫抗性响应机制。结果发现：(1)棉花miR397(ghr miR397)在转录后调控漆酶基因(GhLAC4)的表达,GhLAC4属于蓝铜氧化酶家族,通过调控木质素合成,抵御棉铃虫入侵棉花。(2)GUS报告基因融合表达和酶活性测定表明,ghr miR397在转录后切割靶标基因GhLAC4抑制其表达。(3)利用VIGS(virus induced gene silencing)技术在棉花中沉默和过表达ghr miR397,棉铃虫抗性检测分析表明,沉默miR397表达会增加棉花对棉铃虫的抗性,但过表达ghr miR397则会降低棉花的抗性。(4)选择性和非选择性棉铃虫实验分析、组织化学染色和木质素含量测定表明,沉默GhLAC4表达会减少木质素的积累,增加棉花对棉铃虫的敏感性。研究表明,ghr miR397 GhLAC4模块共同微调棉花木质素合成来参与棉花抗虫性调控,同时也为棉花抗虫育种提供了新思路。     

Response of nutrient dynamics of decomposing pine (Pinus massoniana) needles to simulated N deposition in a disturbed and a rehabilitated forest in tropical China

The effects of simulated N deposition on changes in mass, C, N and P of decomposing pine (Pinus massoniana) needles in a disturbed and a rehabilitated forest in tropical China were studied during a 24-month period. The objective of the study was to test the hypothesis that litter decomposition in a disturbed forest is more sensitive to N deposition rate than litter decomposition in a rehabilitated forest due to the relatively low nutrient status in the former as a result of constant human disturbance (harvesting understory and litter). The litterbag method and N treatments (control, no N addition; low-N, 5 g N m⁻² year⁻¹; medium-N, 10 g N m⁻² year⁻¹) were employed to evaluate decomposition. The results revealed that N addition increased (positive effect) mass loss rate and C release rate but suppressed (negative effect) the release rate of N and P from decomposing needles in both disturbed and rehabilitated forests. The enhanced needle decomposition rate by N addition was significantly related to the reduction in the C/N ratio in decomposing needles. However, N availability is not the sole factor limiting needle decomposition in both disturbed and rehabilitated forests. The positive effect was more sensitive to the N addition rate in the rehabilitated forest than in the disturbed forest, however the reverse was true for the negative effect. These results suggest that nutrient status could be one of the important factors in controlling the response of litter decomposition and its nutrient release to elevated N deposition in reforested ecosystems in the study region.     

红壤中镉在有机酸作用下的解吸行为

采用平衡批处理法,研究了3种有机酸及其两两混合液在序列pH值梯度下（pH 3.0～7.0）对华南山地红壤Cd解吸行为的影响.结果表明,草酸与苹果酸不利于Cd的解吸,反而促进了吸附,其中草酸只是在较高浓度(20 mmol·L^-1)且土壤溶液pH＞5.0时促进解吸.随着pH值升高,草s酸、苹果酸以及不含有机酸的对照溶液对红壤中Cd的解吸率都快速下降.柠檬酸在pH＜5.0时不利于Cd解吸;在pH＞5.0时显著促进Cd解吸,但两种浓度柠檬酸解吸特征有所不同,在低浓度(2 mmol·L^-1)下对镉的解吸率呈降低-升高-降低变化,在高浓度(20 mmol·L^-1)下呈降低-升高变化.在低pH条件下（pH 3.0、4.0）,苹果酸最有利于Cd的解吸,但3种酸对Cd解吸率差别不大,在较高pH条件下(pH 5.0～7.0),柠檬酸最有利于解吸,且解吸率大大高于草酸与苹果酸.有机酸混合没有明显的交互作用,对Cd的解吸率介于相应单独有机酸之间.     

大豆植株不同冠层种子活力及其萌发中抗氧化酶活性

以吉农15号和吉农24号大豆(Glycine max)品种为材料,根据植株高度平均分为下部、中下部、中部、中上部和上部5个冠层,分析不同冠层种子百粒重、种子活力及萌发7天子叶中抗氧化酶活性和丙二醛含量的变化。结果表明,随着植株冠层的升高,大豆百粒重呈先略上升后逐渐下降的趋势,上部冠层种子的百粒重最小。大豆植株下部与中下部冠层种子的发芽率和发芽势较高;并且该两层的种子活力指数显著高于中上部与上部冠层,种子萌发后幼苗的茎粗也显著大于中上部及上部冠层幼苗。下部与中下部冠层种子萌发时子叶中的抗氧化酶活性高于上部,而丙二醛含量低于上部,说明该两层的大豆种子活力高,萌发后幼苗健壮且子叶新陈代谢旺盛,是优选良种的最佳冠层。     

Circulating Tumor Cells Enriched by the Depletion of Leukocytes with Bi-Antibodies in Non-Small Cell Lung Cancer: Potential Clinical Application

Background

It has been considered that the detection methods for circulating tumor cells (CTCs) based on epithelial cell adhesion molecule (EpCAM) underestimate the number of CTCs and may miss a metastatic subpopulation with cancer stem cell (CSC) properties. Therefore, we investigated EpCAM-positive and -negative CTCs in non-small cell lung cancer (NSCLC) patients at different stages, assessed the clinical value of these CTCs and explored their capacity in the following CSC model.

Methods

CTCs were enriched by the depletion of leukocytes with bi-antibodies using a magnetic bead separation technique and then identified by the expression of EpCAM and cytokeratin 7 and 8 using multi-parameter flow cytometry. We determined the distribution of CTCs classified by the expression of EpCAM in 46 NSCLC patients with stages I to IV, assessed the diagnostic value of these CTCs by longitudinal monitoring in 4 index patients during adjuvant therapy and characterized the stemness of these CTCs by the expression of CXCR4 and CD133 in 10 patients.

Results

EpCAM-negative (E-) CTCs were detected to be significantly higher than EpCAM-positive (E+) CTCs in stage IV (p = 0.003). The patients with the percentage of E-CTCs more than 95% (r > 95%) were detected to be significantly increased from 13.3% in stage I-II to 61.1% in stage IV (p = 0.006). Kaplan–Meier analysis indicated that the patients with r > 95% had significantly shorter survival time than those with r ≤ 0.95 (p = 0.041). Longitudinal monitoring of CTCs indicated that the patients with a high percentage of E-CTCs in the blood were not responsive to either chemotherapy or targeted therapy. Further characterization of CTCs revealed that a stem-like subpopulation of CXCR4+CD133+ CTCs were detected to be significantly more prevalent in E-CTCs than that in E+CTCs (p = 0.005).

Conclusions

The enrichment of CTCs by the depletion of leukocytes with bi-antibodies is a valuable method for estimating the number of CTCs, which can be potentially applied in predicting the prognosis, monitoring the therapeutic effect of NSCLC patients and further analyzing the biology of CTCs.