青蒿的遗传转化及青蒿素生物合成的调控

利用发根农杆菌(Agrobacterium rhizogenes)1601，1000，1500，15834，A4，均成功地转化了中药青蒿(Artemisia annua L.)并且建立了pRi1601，pRi15834，pRiA4诱导的发根培养。pRi1601，pRi15834的发根诱导率比其它质粒高。太老或太幼的叶片不利子发根的诱导;发根主要从叶脉的伤口处萌发;带顶芽或带侧芽的叶片容易诱导根，但不一定是发根。光照有利于发根的诱导和发根的生长。以每个发根的“绝对生长速率”(Gtowth Ratio，GR)和绝对“侧根”数量(Number of Side Roots，NSR），通过大量的发根系的筛选，建立了8个发根系，1601-L-1, 1601-L-2, 1601-L-3, 1601-L-4, 15834-L-1, 1601-P-I, 16 01-P-2，15834-L-2。Southern分子检测表明，160l-1-1，1801-L-2, 1601-L-3，1601-L-4，1601-P-1，1601-P-2均为转化子。8个建立的发根系之间无论生长或者QHS的合成存在明显的差异。比较光／暗(16/8hrs)，25℃条件下培养的16 01-L-1，1601-L-2，1601-L-3，1601-L-4，1601-P-l，和1601-P-2，其中16 01-L-3的生长最快，160l-L-1的生长最慢;但是，1601-L-1的QHS的含量最高（可达1. 048%），1601-1-3的QHS的含量最低。160Z-L-3，15834 -L-1和2583:1-L-2的生长速率相差不大。用盛有l000mLMS液体培养基的3000mL的锥形瓶扩大培养1601-L -3，15834-L-1和15834-L-2，转速为ll0rlpm，培养过程中发根容易形成发根球（Hairy Root Balis，HRB），HRB的形成严重影响发根的生长和QHs的合成，HpLC分析表明扩大培养发根中QHS的含量比较低。 改变MS基本培养基中的无机离子的浓度，研究不同无机离子对发根生长和QHS的合成的影响。 l、KN03为18.79×10-3M时有利于1601- L-1生长，为14. 84×10-3M时有利于QHS的合成。NH-4N0-3浓度在10.93-12. 49×10—3M范围内有利于1601-L-1生长，在0-20.62×10-3M范围内对QHS的合成影响不大，大于20. 62×lO-3M不利QHS的合成。培养基中NH-4+／N0-3-比值为0. 37-0. 4-0.52:1时有利于发根的生长，比值为0.52 - 0.58:1时有利于QHS的合成。 2、H-2P0-4-浓度为2.498×10-3M时有利于发根的生长在0-2. 498×l0-3M范围内，随着浓度的提高，促进发根的生长。培养基中的H2P4 -的浓度在0-1.249×lO-3M的范围内，随着浓度的提高，促进QHS的合成，为1.249×10-3M时QHS的含量最高。 3、培养基中最适16 01-L-1生长的Ca-2+浓度为0.198- 0.766×10-3M，大于或小于该浓度范围，显著地抑制发根的生长。但是，在0-3.695×10-3M范围内，随着培养基中Ca-2+浓度提高，促进QHS的合成，最适Ca-2+浓度为3.695×l0-3M。 4、培养基中不加Mg-2+时，完全抑制发根生长，在0. 142×10-3M-7.506×l0-3M浓度范围内，对发根生长影响没有明显的差别。但是，HPLC和UV分析发根中QHS含量，培养基中不加Mg-2+时，发根中QHS含量最高。 5、培养基中的Fe-2+浓度在0. 25 -1．0×10-3M范围内，同时有利于16 01- L-1的生长和QHS的形成。 6、培养基中最适合予16 01- L-3生长的KI浓度为2.5ppm，大于或小予该浓度均显著地抑制发根的生长，培养基中加入KI明显地降低发根中的QHS的含量。 7、H2BO3对l601-L-l生长影响不大，HPLC分析QHS的含量，培养基中的H3BO3浓度为100ppm和400ppm，QHS的含量分别为1.69mg/g和1.80mg/g(DW)。 8、Cu-2+对1601-L-3的生长影响显著，最适合1601-L-3生长的Cu-2+浓度为1.00ppm，在0 -1.00ppm的浓度范围内，随着培养基中的Cu+浓度的提高，发根的生物量不断增加。培养基中QHS合成的最适Cu2+浓度为0.05ppm，大于或小于该浓度均显著地抑制发根中QHS的合成。 比较光培养和暗培养对发根生长的影响，结果表明光照明显地促进1601-L-l的生长，暗培养明显不利于发根的生长。最适合于发根生长的温度为25℃，大于35℃显著地抑制发根的生长，影响发根的根尖细胞的正常分裂。 改变培养基中的蔗糖浓度和在发根培养的不同时期给培养基中添加蔗糖，试验结果表明蔗糖作为碳源对1601-L-3和1601-L-1的生长具有显著的影响。 (1)培养基中缺少蔗糖显著地抑制发根的生长。 (2)发根培养的前5天时间内，蔗糖浓度为30- 60glL昀培养基最有利于发根的生长，50glL的培养基中的发根生长最快，培养基中的蔗糖浓度大于60g/L小于30g／L时，发根的生物量增加较少。 (3)发根培养至第15天时，蔗糖浓度为60g/L的培养基最有利予发根的生物量的增加。发根培养至30天时，蔗糖浓度为60-90g/L的培养基，发根的生物量的增加相差不大，但是为蔗糖浓度为30-40g/L的培养基中的发根生物量一倍。 (4)发根培养过程中，分别于第5和15天给蔗糖浓度为30g/L的培养基中添加一次或二次蔗糖，使培养基中的蔗糖终浓度相当于60g/L或90g/L，培养至30天时，添加蔗糖的培养基中的发根的干重生物量相当于不添加蔗糖培养基中的发根生物量一倍，相当于初始蔗糖浓度为60g/L和90g/L培养基中发根的生物量。 (5)随着培养基中蔗糖浓度的提高，发根干重/鲜重比显著增加。培养基中的蔗糖的消耗量与发根生物量的增加呈正相关，蔗糖消耗越多，发根生物量的增加越大。 比较pH值对发根生长和QHS合成的影响表明，灭菌前pH值在5.O-6.5范围内的培养基适合予1601-L-1的生长，小于5.O不利于发根的生长，pH5.8有利于1601-1-1生长和QHS的生物合成。发根收获时培养基中的pH值一般为4.5-5.2. pH7.O抑制发根的生长，pHl0.O对发根具有强烈的致死作用。发根在培养过程中，对培养基中的pH值具有显著的调节作用，发根能在很短的时间内(24- 48hrs)使pl:l值为5.8、6.4、7.0培养基降低到pH4. 5-5.2，pH为5.8的培养基有利于QHS合成。 比较不同基本培养基对发根生长和QHS合成的影响，试验结果表明N6、DCR、Litvay培养基有利于1601-L-1的生长，WS、White、B5培养基不利于发根的生长。DCR培养基中的QHS含量最高。 根据三水平试验选用三水平正交表来安排试验的原则，选用三水平正交表L7(3-)，研究多因子效应对发根生长和QHS合成的影响，试验结果表明，Mg2+，Fe2+，Mn-2+，NH4NO3，KN03 ,KI,Ca-2+为发根生长的主要因子，NH4N03，KNOs，Mg2+，Ca2+，肌醇为QHS合成的主要因子。 通过TLC分析发根中QHS和其它化学成分，同时比较发根和无菌苗及野生植株的化学成分，发根和无菌苗均能合成包括QHS在内的野生青蒿叶片中的大部分非挥发性的化台 物。 研究青蒿植株在发育过程中QHS的含量的变化以及发根、无菌苗和野生青蒿中QHS的合成，HP分析结果表明，l、不同的单株青蒿之间的QHS量相差很大。2、同一植株幼 叶的QHS含量比老叶的QHS含量高。3、不同单株青蒿之间达到最高QHS含量的时间不一样，开花期或开花之前。4、无菌苗（带根）或者不带根丛生芽均能合成QHS，但是带根的无菌蕾的QHS量比丛生芽中的QIS的含量高。5、不同发根农杆菌转化的发根系1601-L-1和15834-L-1都能合成ＱＨＳ。

基于GIS与神经网络的黄土丘陵区土壤水分模型研究

土壤水分不足是黄土高原丘陵区植被建设的主要限制因子,土壤水分的空间分布受外界气象因子、土地利用与复杂地形等的影响,关系比较复杂。本研究利用黄土丘陵区纸坊沟流域的土壤水分试验资料,建立了基于GIS的BP神经网络模型,模型中同时考虑了多个因子对土壤水分空间分布的影响,利用实测资料对网络进行训练后对整个流域进行了预测,预测结果与实际情况较为一致,表明应用GIS与BP神经网络研究区域复杂地形下的土壤水分分布规律是可行的。

Insulin-like growth factor-binding protein-3 plays an important role in regulating pharyngeal skeleton and inner ear formation and differentiation

Insulin-like growth factor-binding protein (IGFBP)-3 is the major insulin-like growth factor (IGF) carrier protein in the bloodstream. IGFBP-3 prolongs the half-life of circulating IGFs and prevents their potential hypo-glycemic effect. IGFBP-3 is also expressed in many peripheral tissues in fetal and adult stages. In vitro, IGFBP-3 can inhibit or potentiate IGF actions and even possesses IGF-independent activities, suggesting that local IGFBP-3 may also have paracrine/autocrine function(s). The in vivo function of IGFBP-3, however, is unclear. In this study, we elucidate the developmental role of IGFBP-3 using the zebrafish model. IGFBP-3 mRNA expression is first detected in the migrating cranial neural crest cells and subsequently in pharyngeal arches in zebrafish embryos. IGFBP-3 mRNA is also persistently expressed in the developing inner ears. To determine the role of IGFBP-3 in these tissues, we ablated the IGFBP-3 gene product using morpholino-modified antisense oligonucleotides (MOs). The IGFBP-3 knocked down embryos had delayed pharyngeal skeleton morphogenesis and greatly reduced pharyngeal cartilage differentiation. Knockdown of IGFBP-3 also significantly decreased inner ear size and disrupted hair cell differentiation and semicircular canal formation. Furthermore, reintroduction of a MO-resistant form of IGFBP-3 "rescued" the MO-induced defects. These findings suggest that IGFBP-3 plays an important role in regulating pharyngeal cartilage and inner car development and growth in zebrafish.

Multiple Axial Cracks in A Coated Hollow Cylinder due to Thermal Shock

The transient thermal stress problem of an inner-surface-coated hollow cylinder with multiple pre-existing surface cracks contained in the coating is considered. The transient temperature, induced thermal stress, and the crack tip stress intensity factor (SIF) are calculated for the cylinder via finite element method (FEM), which is exposed to convective cooling from the inner surface. As an example, the material pair of a chromium coating and an underlying steel substrate 30CrNi2MoVA is particularly evaluated. Numerical results are obtained for the stress intensity factors as a function of normalized quantities such as time, crack length, convection severity, material constants and crack spacing. (c) 2005 Elsevier Ltd. All rights reserved.

“Deborah Numbers”, Coupling Multiple Space and Time Scales and Governing Damage Evolution to Failure

Two different spatial levels are involved concerning damage accumulation to eventual failure. nucleation and growth rates of microdamage nN* and V*. It is found that the trans-scale length ratio c*/L does not directly affect the process. Instead, two independent dimensionless numbers: the trans-scale one * * ( V*)including the * **5 * N c V including mesoscopic parameters only, play the key role in the process of damage accumulation to failure. The above implies that there are three time scales involved in the process: the macroscopic imposed time scale tim = /a and two meso-scopic time scales, nucleation and growth of damage, (* *4) N N t =1 n c and tV=c*/V*. Clearly, the dimensionless number De*=tV/tim refers to the ratio of microdamage growth time scale over the macroscopically imposed time scale. So, analogous to the definition of Deborah number as the ratio of relaxation time over external one in rheology. Let De be the imposed Deborah number while De represents the competition and coupling between the microdamage growth and the macroscopically imposed wave loading. In stress-wave induced tensile failure (spallation) De* < 1, this means that microdamage has enough time to grow during the macroscopic wave loading. Thus, the microdamage growth appears to be the predominate mechanism governing the failure. Moreover, the dimensionless number D* = tV/tN characterizes the ratio of two intrinsic mesoscopic time scales: growth over nucleation. Similarly let D be the “intrinsic Deborah number”. Both time scales are relevant to intrinsic relaxation rather than imposed one. Furthermore, the intrinsic Deborah number D* implies a certain characteristic damage. In particular, it is derived that D* is a proper indicator of macroscopic critical damage to damage localization, like D* ∼ (10–3~10–2) in spallation. More importantly, we found that this small intrinsic Deborah number D* indicates the energy partition of microdamage dissipation over bulk plastic work. This explains why spallation can not be formulated by macroscopic energy criterion and must be treated by multi-scale analysis.

A combined statistical model for multiple motifs search

Transcription factor binding sites (TFBS) play key roles in genebior 6.8 wavelet expression and regulation. They are short sequence segments with de¯nite structure and can be recognized by the corresponding transcription factors correctly. From the viewpoint of statistics, the candidates of TFBS should be quite di®erent from the segments that are randomly combined together by nucleotide. This paper proposes a combined statistical model for ¯nding over- represented short sequence segments in di®erent kinds of data set. While the over-represented short sequence segment is described by position weight matrix, the nucleotide distribution at most sites of the segment should be far from the background nucleotide distribution. The central idea of this approach is to search for such kind of signals. This algorithm is tested on 3 data sets, including binding sites data set of cyclic AMP receptor protein in E.coli, PlantProm DB which is a non-redundant collection of proximal promoter sequences from di®erent species, collection of the intergenic sequences of the whole genome of E.Coli. Even though the complexity of these three data sets is quite di®erent, the results show that this model is rather general and sensible.

Specklegram in a multiple-mode fiber and its dependence on longitudinal modes of the laser source

A specklegram in a multimode fiber (MMF) has successfully been used as a sensor for detecting external disturbance. Our experiments showed that the sensitivity in the sensor with a multiple longitudinal-mode laser as its source was much higher than that with a single longitudinal-mode laser. In addition, the near-field pattern observations indicated that the coupling between different transverse modes in the MMF is quite weak. Based on the experimental results, a theoretical model for the speckle formation is proposed, taking a bend-caused phase factor into consideration. It is shown in the theoretical analysis that the interferences between different longitudinal modes make a larger contribution to the specklegram signals. (C) 2007 Optical Society of America.

Hepatocyte growth factor enhances the generation of high-purity oligodendrocytes from human embryonic stem cells

Generation of homogeneous oligodendrocytes as donor cells is essential for human embryonic stem cell (hESC)-based cell therapy for demylinating diseases. Herein we present a novel method for efficiently obtaining mature oligodendrocytes from hESCs with high purity (79.7 +/- 6.9%), using hepatocyte growth factor (HGF) and G5 supplement(containing insulin, transferrin, selenite, biotin, hydrocortisone, basic fibroblast growth factor and epidermal growth factor) in a four-step method. We induced hESCs into neural progenitors (NP) with HGF (5 ng/ml) and G5 (1 x) supplemented medium in an adherent differentiation system. The purified NPs were amplified in suspension as neurospheres for 1 month, and terminal oligodendrocyte differentiation was then induced by G5 supplement withdrawal and HGF treatment (20 ng/ml). The cells generated displayed typical morphologies of mature oligodendrocytes and expressed oligodendrocyte markers O4 and myelin basic protein (MBP). Our result revealed that HGF significantly enhanced the proliferation of hESC-derived NPs and promoted the differentiation as well as the maturation of oligodendrocytes from NPs. Further studies suggest that HGF/c-Met signaling pathway might play an important role in oligodendrocyte differentiation in our system. Our studies provide a means for generating the clinically relevant cell type and a platform for deciphering the molecular mechanisms that control oligodendrocyte differentiation. (C) 2009 International Society of Differentiation. Published by Elsevier Ltd. All rights reserved.

Immunolocalization and expression of vascular endothelial growth factor receptors (VEGFRs) and neuropilins (NRPs) on Keratinocytes in human epidermis

Vascular endothelial growth factor (VEGF) plays an important role in normal and pathological angiogenesis. VEGF receptors (VEGFRs, including VEGFR-1, VEGFR-2, and VEGFR-3) and neuropilins (NRPs, including NRP-1 and NRF-2) are high-affinity receptors for V

Expression of VEGFR-2 on HaCaT cells is regulated by VEGF and plays an active role in mediating VEGF induced effects

Vascular endothelial growth factor (VEGF) and its receptor VEGFR-2 play important roles in mitogenesis and chemotaxis of endothelial cells. In normal human skin, VEGF is expressed and secreted by epidermal keratinocytes. Emerging data suggest that keratin

Elongation Factor ELL (Eleven-Nineteen Lysine-rich Leukemia) Acts as a Transcription Factor for Direct Thrombospondin-1 Regulation

The eleven-nineteen lysine-rich leukemia (ELL) gene undergoes translocation and fuses in-frame to the multiple lineage leukemia gene in a substantial proportion of patients suffering from acute forms of leukemia. Studies show that ELL indirectly modulates transcription by serving as a regulator for transcriptional elongation as well as for p53, U19/Eaf2, and steroid receptor activities. Our in vitro and in vivo data demonstrate that ELL could also serve as a transcriptional factor to directly induce transcription of the thrombospondin-1 (TSP-1) gene. Experiments using ELL deletion mutants established that full-length ELL is required for the TSP-1 up-regulation and that the trans-activation domain likely resides in the carboxyl terminus. Moreover, the DNA binding domain may localize to the first 45 amino acids of ELL. Not surprisingly, multiple lineage leukemia-ELL, which lacks these amino acids, did not induce expression from the TSP-1 promoter. In addition, the ELL core-response element appears to localize in the -1426 to -1418 region of the TSP-1 promoter. Finally, studies using zebrafish confirmed that ELL regulates TSP-1 mRNA expression in vivo, and ELL could inhibit zebrafish vasculogenesis, at least in part, through up-regulating TSP-1. Given the importance of TSP-1 as an anti-angiogenic protein, our findings may have important ramifications for better understanding cancer.

Zebrafish peptidoglycan recognition protein SC (zfPGRP-SC) mediates multiple intracellular signaling pathways

Insect PGRPs can function as bacterial recognition molecules triggering proteolytic and/or signal transduction pathways, with the resultant production of antimicrobial peptides. To explore if zebrafish peptidoglycan recognition protein SC (zfPGRP-SC) has such effects, RNA interference (siRNA) and high-density oligonucleotide microarray analysis were used to identify differentially expressed genes regulated by zfPGRP-SC. The mRNA levels for a set of genes involved in Toll-like receptor signaling pathway, such as TLRs, SARM, MyD88, TRAF6 and nuclear factor (NF)-kappa B2 (p100/p52), were examined by quantitative RT-PCR (QT-PCR). The results from the arrays and QT-PCR showed that the expression of 133 genes was involved in signal transduction pathways, which included Toll-like receptor signaling, Wnt signaling, BMP signaling, insulin receptor signaling, TGF-beta signaling, GPCR signaling, small GTPase signaling, second-messenger-mediated signaling, MAPK signaling, JAK/STAT signaling, apoptosis and anti-apoptosis signaling and other signaling cascades. These signaling pathways may connect with each other to form a complex network to regulate not just immune responses but also other processes such as development and apoptosis. When transiently over-expressed in HEK293T cells, zfPGRP-SC inhibited NF-kappa B activity with and without lipopolysacharide (LPS) stimulation. (C) 2008 Elsevier Ltd. All rights reserved.

Food web of benthic macro invertebrates in a large Yangtze River-connected lake: the role of flood disturbance

This Study was conducted in Lake Dongtinghu, a large river-connected lake on the Yangtze River flood-plain, China. Our goal was to determine trophic relationships among benthic macroinvertebrates, as well as the effects of flood disturbance on the benthic food web of a river-connected lake. Macroinvertebrates in the lake fed mainly on detritus and plankton (both zooplankton and phytoplankton). Food web Structure in Lake Dongtinghu was characterized by molluscs as the dominant group, low connectance, high level of omnivory. based oil detritus and primary production, and most ingestion concentrating on a few links. Our analyses showed that flood disturbance is an important factor affecting the benthic food web in Lake Dongtinghu. The numbers of species and functional feeding groups (FFGs), and the density and biomass of macroinvertebrates decreased significantly during flooding. Connectance was higher during the flood season than in other seasons, indicating that floods have a strong effect on connectance in this Yangtze River-connected lake. Flood effects on the benthic web were also evident in the decrease of niche overlaps within and anion, FFGs. Our results provide useful information regarding biodiversity conservation on the Yangtze floodplain. Reconstructing and maintaining natural and regular flow regimes between Yangtze lakes and the river is essential for restoration of macroinvertebrates on the floodplain.

U19/Eaf2 knockout causes lung adenocarcinoma, B-cell lymphoma, hepatocellular carcinoma and prostatic intraepithelial neoplasia

Upregulated gene 19 (U19)/ELL-associated factor 2 (Eaf2) is a potential human tumor suppressor that exhibits frequent allelic loss and downregulation in high-grade prostate cancer. U19/Eaf2, along with its homolog Eaf1, has been reported to regulate transcriptional elongation via interaction with the eleven-nineteen lysine-rich leukemia (ELL) family of proteins. To further explore the tumor-suppressive effects of U19/Eaf2, we constructed and characterized a murine U19/Eaf2-knockout model. Homozygous or heterozygous deletion of U19/Eaf2 resulted in high rates of lung adenocarcinoma, B-cell lymphoma, hepato cellular carcinoma and prostate intraepithelial neoplasia. Within the mouse prostate, U19/Eaf2 defficiency enhanced cell proliferation and increased epithelial cell size. The knockout mice also exhibited cardiac cell hypertrophy. These data indicate a role for U19/Eaf2 in growth suppression and cell size control as well as argue for U19/Eaf2 as a novel tumor suppressor in multiple mouse tissues. The U19/Eaf2 knockout mouse also provides a unique animal model for three important cancers: lung adenocarcinoma, B-cell lymphoma and hepatocellular carcinoma.

Molecular evolution of connective tissue growth factor in Cyprinidae (Teleostei : Cypriniformes)

Connective tissue growth factor (CTGF) plays an important role in regulation of cell growth, differentiation, apoptosis and individual development in animals. The study of sequences variation and molecular evolution of CTGF gene across various species of the cyprinid could be helpful for understanding of speciation and gene divergence in this kind of fish. In this study, 19 novel sequences of CTGF gene were obtained from the representative species of the family Cyprinidae using PCR amplification, cloning and sequencing. Phylogenetic relationships of Cyprinidae were reconstructed by neighbor-joining (NJ), maximum parsimony (MP), maximum likelihood (ML) and Bayesian method. Oryzias latipes from the family Cyprinodontidae was assigned to be the outgroup taxon. Leuciscini and Barbini were clustered into the monophyletic lineages, respectively, with the high nodal supports. The estimation of the ratio of non-synonymous to synonymous substitution (dN/dS) for the various branches indicated that there stood the different evolution rates between the Leuciscini and the Barbini. With the ratio of dN/dS of the Leuciscini being lower than that of the Barbini, species within the Barbini were demonstrated to be subjected to the relatively less selection pressure and under the relaxable evolution background. A 6 by indel (insertion/deletion) was found at the 5' end of CTGF gene of Cyprinidae, and this 6 by deletion only appeared in the Leuciscini, which is a typical characteristic of the Leuciscini and provides evidence for the monophylogeny of the Leuciscini. For the amino acid sequences of CTGF protein, the most variations and indels were distributed in the signal region and IGFBP region of this protein, implying that these variations were correlated with the regulation of the CTGF gene expression and protein activity. (c) 2007 National Natural Science Foundation of China and Chinese Academy of Sciences. Published by Elsevier Limited and Science in China Press. All rights reserved.