茭白茎部CTK及ABA含量动态变化分析

通过比较分析正常茭白与灰茭两种膨大表型茎部发育期间的CTK和ABA含量的动态变化,探索茭白茎部菰黑粉菌生长分布与CTK和ABA含量变化的调节关系.本实验以灰茭和正常茭不同发育时期茎部为实验材料,并采用酶联免疫吸附分析法(简称ELISA)测定实验材料内CTK和ABA的含量,结果表明,灰茭茎部CTK含量峰值出现在孢子形成期,可能与灰孢子的增殖有关,而正常茭出现在分蘖期,可能与茭白组织不断分裂有关;ABA激素在灰茭茎部含量一直较高,在孢子形成期后期达到峰值,正常茭在8叶期和膨大期较高,内源激素ABA的高水平表达可能是由菰黑粉菌侵染茭白以及大量繁殖引起的.总体上CTK和ABA在灰茭和正常茭白内的关系不是单一的,而是相辅相成的.     

菰黑粉菌孢子萌发过程形态学观察及系统发育研究

通过显微观察法研究菰黑粉菌厚垣孢子在28℃、PDA液体培养基中静止萌发各时期的菌体形态,克隆了菰黑粉菌18S rDNA序列并测序,结合已有ITS-5.8S rDNA序列和18S rDNA序列分析其系统发育地位.结果表明,从厚垣孢子到形成稳定的担孢子需要62~72 h,经历厚垣孢子萌发、芽管形成、先菌丝形成、担孢子和小孢子等几个阶段,在体外培养时多以棒状担孢子形态存在,培养30 d后,在培养基表面或边缘形成白色丝状菌丝;系统发育分析结果显示菰黑粉菌与Ustilago属和Sporisorium属亲缘关系较近,与黑粉菌属中的Ustilago triodiae亲缘关系最近.     

茭白黑粉菌的分离鉴定研究

以"余茭4号"灰茭为材料,进行了茭白黑粉菌的分离,并进行了ITS-5.8S rDNA的分子序列测定。结果表明:ITS-5.8S rDNA扩增片段大小775bp,通过DNA Star软件进行比较分析,结果表明:分离出的黑粉菌(Ustilago esculenta)是黑粉菌属的边缘物种。     

低温下茭白品种龙茭2号细胞膜损伤与冷胁迫分析

以浙江省主栽茭白品种浙茭911为对照,比较它与新品种龙茭2号在低温下叶片丙二醛含量、相对电导率的变化以及冷胁迫指数和耐寒性.结果表明,龙茭2号在低温,尤其是持续低温下,电导率变化小,丙二醛含量少,对寒冷适应较快,恢复能力较强.两个茭白品种的冷胁迫指数只有在5℃的恢复阶段存在显著差异,且龙茭2号的茎和叶冷胁迫指数都较低,干物质积累受寒冷胁迫影响小,具有较强的耐寒性.     

热变形镁合金退火显微组织的定量研究

对热变形AZ31镁合金的显微组织、晶粒尺寸分布、平均晶粒尺寸、再结晶晶粒数目以及变形织构随退火时间的变化进行了定量分析,研究了不同热变形量AZ31镁合金在503 K的等温退火行为。结果表明:热变形AZ31镁合金的细晶组分随着退火时间的延长不断降低,退火过程按退火温度可分为孕育、再结晶急速长大和晶粒正常长大三个阶段,且各阶段的其长短几乎不受变形程度的影响。变形形成的微观织构在整个退火过程中几乎没有变化,说明热变形镁合金在退火过程中没有新核形成,即为连续静态再结晶。     

茭白品种间基因组ISSR多态性分析

对浙江、湖北、江苏等省3个野生茭白材料17个不同品种的茭白种质资源进行了收集,利用ISSR方法对茭白植株进行了基因组多态性分析.结果表明,野生茭白与栽培茭白品种间具有明显的多态性,不同来源的栽培茭白品种间也具有一定的多态性,多态性比例分布在12%~25%之间.利用DPS软件对茭白植株的多态性位点进行的系统聚类分析发现,野生茭白与栽培茭白间距离较远,茭白地域分布及栽培特性与种质资源多样性相关.     

A549细胞中Dectin-1受体在烟曲霉刺激后的表达规律

为明确烟曲霉感染肺上皮细胞过程中宿主与病原菌烟曲霉之间的相互作用,该研究探究在烟曲霉孢子内化侵入肺上皮细胞过程中细胞表面dectin-1受体的表达随时间的变化规律及其与侵入孢子的空间位置关系。通过RT-PCR的方法检测烟曲霉孢子刺激Ⅱ型肺上皮细胞A549后不同时间的m RNA水平变化,以及利用荧光显微镜观察烟曲霉孢子侵入A549细胞后其周围dectin-1的分布情况。结果表明:在该侵入过程中,A549细胞中dectin-1的m RNA表达水平无显著性变化,但其可在孢子周围形成"吞噬环"。因此,在烟曲霉内化侵入肺上皮细胞的过程中,dectin-1的表达不发生变化,这与人支气管上皮细胞中dectin-1的诱导型表达增加现象不同;但在烟曲霉孢子周围,dectin-1发生聚集,这种模式识别受体在病原体周围的聚集可能会促进吞噬。     

共沉淀法合成小粒径PDP绿粉中的物相转变和颗粒生长过程研究

使用化学共沉淀法合成出粒径为1～2μm的Mg掺杂的PDP用铝酸盐绿色荧光粉,研究了共沉淀产物在高温煅烧中的物相变化和颗粒长大过程,并使用该超细PDP绿粉进行了涂屏实验.结果表明,共沉淀产物的物相转变过程异常复杂,中间相包括Ba-C3、γ-Al2O3、BaF2、BaAl2O4和两个含Mn的化合物,在整个物相转变过程中没有...     

灰口铸铁在冲击高压下的变化——Ⅰ、石墨→金刚石相变

在冲击高压下,工业用铁碳合金中的稳定和亚稳定相会发生一系列变化。本篇中笔者研究灰口铸铁在冲击高压下石墨转化为金刚石的相变。通过冲击试验数据,提出了在灰口铸铁中形成金刚石相的条件;用扫描电镜观察了金刚石的形貌;用扫描电子探针分析仪对金刚石的碳成分进行了波谱线扫描分析;对灰口铸铁中形成的金刚石结构及其性能进行了分析讨论。     

高铝锌合金凝固过程中先共晶富铝相的二次形核与晶粒合并现象

通过定向凝固-液淬试验和金相定量分析,研究了二元ZA28.4合金的微观凝固特性.研究表明:高铝锌合金的先共晶富铝相有二次形核现象,二次形核的倾向性随冷却速度的增大先增强后减弱;初生α相和铸态晶粒的数目随冷却速度增大而增大.     

Nuclear localization of aminoacyl-tRNA synthetases using single-cell capillary electrophoresis laser-induced fluorescence analysis

Aminoacyl-tRNA synthetases (aaRSs) are a family of enzymes whose function in specific aminoacylation of tRNAs is central to the process of protein translation, which occurs in the cytoplasm of all living cells. In addition to their well-established cytoplasmic localization, fluorescence microscopy studies and analysis of the aminoacylation state of nuclear tRNAs have revealed that synthetases are localized in the nuclei of cells from several species including Xenopus laevis and Saccharomyces cerevisiae. Whether nuclear localization of aaRSs is a general phenomenon that occurs in all eukaryotic cells is an open question. In the work described here, human methionyl-tRNA synthetase (MRS) and human lysyl-tRNA synthetase (KRS) were expressed in human-derived DeltaH2-1 osteosarcoma cells as enhanced green fluorescent protein (EGFP) fusion proteins. The subcellular localization of these EGFP-aaRSs was first probed by fluorescence microscopy using cells that coexpressed EGFP-aaRS and a nuclear marker fusion protein, nuDsRed. As expected, both aaRSs were present in the cytosol, while only EGFP-MRS was also clearly localized in the nucleus. To confirm these findings, and to investigate a potentially more sensitive, general method for nuclear localization studies, capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection was used to analyze single DeltaH2-1 cells expressing both EGFP-aaRS and nuDsRed. While cytosolic EGFP signals were detected for both EGFP-MRS and EGFP-KRS, only EGFP-MRS was found in the nucleus, along with nuDsRed. The detection of EGFP-MRS in nuclei of DeltaH2-1 cells demonstrates the feasibility of using CE-LIF analysis in nuclear localization studies of proteins in mammalian cells.     

Measuring the doxorubicin content of single nuclei by micellar electrokinetic capillary chromatography with laser-induced fluorescence detection

Individual nuclei isolated from the human leukemia CCRF-CEM and CEM-C2 cells treated with doxorubicin (DOX) were in-column lysed with a sodium dodecyl sulfate (SDS) containing buffer, their contents were then separated by micellar electrokinetic capillary chromatography using the same lysing buffer, and the DOX content was detected by laser-induced fluorescence. Use of a microscope for the selection of one nucleus from the nuclear preparation decreases the possibility of introduction of other subcellular components that are commonly found as impurities in subcellular fractions. The presence of SDS in the running buffer made negligible the DNA's quenching effect on DOX fluorescence, which often compromises quantification of DOX by direct imaging, making it possible to carry out the first direct measurement of the doxorubicin content of isolated nuclei. On average, nuclei from CCRF-CEM and CEM/C2 cell lines contained 85 +/-64 (n = 6) and 91 +/- 51 (n =7) amol of DOX, respectively. These values correspond to 74 and 65% of the average total cellular content as determined by single-cell analysis of the corresponding cell types. It is envisioned that this approach could become an important bioanalytical tool to investigate the effect of treatments with fluorescent drugs targeting the nucleus.     

Detection of quadruplex DNA structures in human telomeres by a fluorescent carbazole derivative

Single-stranded telomeric DNA tends to form a four-base-paired planar structure termed G-quadruplex. This structure was easily formed in vitro in the presence of monovalent cations. However, the existence of this structure in native human telomeres is unclear. Here we address this important question through the distinctive properties of 3,6-bis(1-methyl-4-vinylpyridinium)carbazole diiodide (BMVC) upon binding to various DNA structures. Although the fluorescence of BMVC increases significantly in the presence of DNA, BMVC has high sensitivity and binding preference to quadruplex d(T(2)AG(3))(4) over duplex DNA. In addition, the fluorescent emissions were characterized around 575 nm for quadruplex d(T(2)AG(3))(4) and 545 nm for most of duplex DNA. The 575-nm fluorescence emissions were detected in the mixtures of 2 nM BMVC with the chromosomal DNA that were extracted from human cells, suggesting the presence of quadruplex structure in human nucleus. Further analyzing the BMVC fluorescence at the ends of metaphase chromosomes and other regions of chromosomes, we detected the quadruplex-binding BMVC fluorescence at telomere-proximal regions. Together these results provide the first evidence for the presence of quadruplex structures in human telomeres.     

Permeability of the nuclear envelope at isolated Xenopus oocyte nuclei studied by scanning electrochemical microscopy

In interphase eukaryotic cells, molecular transport between the cytoplasm and the nucleus is mediated by the nuclear pore complex (NPC), which perforates the double-membraned nuclear envelope (NE). Local permeability of the NE at large intact nuclei (approximately 400 microm in diameter) isolated from Xenopus laevis oocytes was studied by scanning electrochemical microscopy (SECM). Steady-state tip current versus tip-nucleus distance curves (approach curves) were measured with 10- and 2-microm-diameter Pt disk microelectrodes at the nuclei in isotonic buffer solutions containing redox-active molecules. The approach curves in the normalized form are independent of the tip diameter, indicating diffusion-limited membrane transport of the redox molecules. SECM chronoamperometry demonstrated that a decrease in the steady-state tip current at short tip-nucleus distances is due to smaller diffusion coefficients and concentrations of the redox molecules in the nucleus than those in the buffer solution. The experimental approach curves fit very well with theoretical ones for freely permeable membranes, yielding the NE permeability to the molecules that is at least 2 orders of magnitude larger than permeability of bilayer lipid membranes and cell membranes. This result indicates that passive transport of the redox molecules across the NE is facilitated by open NPC pores. The flux of the redox molecules sustainable by a single NPC channel (>9.8 x 10(6) molecules per NPC per second) and the diameter of the channel pore (>15 nm) were estimated from the SECM data by assuming the NE as an array of nanometer-sized NPC pores. The effects of the redox molecules on the nucleus and the NPC function were examined by studying signal-mediated nuclear import of rhodamine-labeled bovine serum albumin with and without nuclear localization signals by fluorescence microscopy.     

Pluronic F127 nanomicelles engineered with nuclear localized functionality for targeted drug delivery

PKKKRKV (Pro-Lys-Lys-Lys-Arg-Lys-Val, PV7), a seven amino acid peptide, has emerged as one of the primary nuclear localization signals that can be targeted into cell nucleus via the nuclear import machinery. Taking advantage of chemical diversity and biological activities of this short peptide sequence, in this study, Pluronic F127 nanomicelles engineered with nuclear localized functionality were successfully developed for intracellular drug delivery. These nanomicelles with the size ~ 100 nm were self-assembled from F127 polymer that was flanked with two PV7 sequences at its both terminal ends. Hydrophobic anticancer drug doxorubicin (DOX) with inherent fluorescence was chosen as the model drug, which was found to be efficiently encapsulated into nanomicelles with the encapsulation efficiency at 72.68%. In comparison with the non-functionalized namomicelles, the microscopic observation reveals that PV7 functionalized nanomicelles display a higher cellular uptake, especially into the nucleus of HepG2 cells, due to the nuclear localization signal effects. Both cytotoxicity and apoptosis studies show that the DOX-loaded nanomicelles were more potent than drug nanomicelles without nuclear targeting functionality. It was thus concluded that PV7 functionalized nanomicelles could be a potentially alternative vehicle for nuclear targeting drug delivery.     

用荧光原位杂交(FISH)技术鉴定赤潮甲藻的研究

探索了用荧光原位杂交（Fluorescence In Situ Hybridization,FISH）技术检测赤潮藻的可行性。用FISH技术鉴定了2株已知的赤潮甲藻——东海原甲藻（Prorocentrum donghaiense）和塔马亚历山大藻（Alexandrium tamarense）。荧光标记的DNA探针是东海原甲藻的核糖体转录单元内间隔区（ITS）序列。研究结果如下：（1）在荧光显微镜下观察与荧光探针杂交后的东海原甲藻细胞,在细胞顶端可见绿色的荧光,而作为对照的塔马亚历山大藻在与标记的探针杂交后没有产生绿色荧光,说明探针只与目标株东海原甲藻反应,不与对照组反应,表明根据ITS序列设计的探针是高度特异的;（2）材料固定后,经蓝光激发,均未产生细胞内源的叶绿素和甲藻素等色素荧光干扰杂交信号的现象,说明固定材料的方法正确。以上的结果表明,FISH技术在鉴定赤潮微藻方面具有其它技术无法比拟的优越性：准确、快速、简单,将其应用到现场定性、定量检测赤潮微藻方面将具有很大的潜力。     

Altered Actin Dynamics and Functions of Osteoblast-Like Cells in Parabolic Flight may Involve ERK1/2

Osteoblasts are sensitive to mechanical stressors such as gravity and alter their cytoskeletons and functions to adapt; however, the contribution of gravity to this phenomenon is not well understood. In this study, we investigated the effects of acute gravitational changes on the structure and function of osteoblast ROS17/2.8 as generated by parabolic flight. The changes in microfilament cytoskeleton was observed by immunofluorescence stain of Texas red conjugated Phalloidin and Alexa Fluor 488 conjugated DNase I for F-actin and G-actin, respectively. To examine osteoblast function, ALP (alkaline phosphatase) activity, osteocalcin secretions and the expression of ALP, COL1A1 (collagen type I alpha 1 chain) and osteocalcin were detected by modified Gomori methods, radioimmunity and RT-PCR, respectively. Double fluorescence staining of phosphorylated p44/42 and F-actin were performed to observe their colocalization relationship. The established semi-quantitative analysis method of fluorescence intensity of EGFP was used to detect the activity changes of COL1A1 promoter in EGFP-ROS cells with MAPK inhibitor PD98059 or F-actin inhibitor cytochalasin B. Results indicate that the altered gravity induced the reorganization of microfilament cytoskeletons of osteoblasts. After 3 h parabolic flight, F-actin of osteoblast cytoskeleton became thicker and directivity, whereas G-actin shrunk and became more concentrated at the edge of nucleus. The excretion of osteocalcin, the activity of ALP and the expression of mRNA decreased. Colocalization analysis indicated that phosphorylated p44/42 MAPK was coupled with F-actin. Inhibitor PD98059 and cytochalasin B decreased the fluorescence intensity of EGFP-ROS cells. Above results suggest that short time gravity variations induce the adjustment of osteoblast structure and functional and ERK1/2 signaling maybe involve these responses. We believe that it is an adaptive method of the osteoblasts to gravity alteration that structure alteration inhibits the function performing.     

Green-emissive carbon quantum dots with high fluorescence quantum yield: Preparation and cell imaging

High fluorescence quantum yield (QY), excellent fluorescence stability, and low toxicity are essential for a good cellular imaging fluorescent probe. Green-emissive carbon quantum dots (CQDs) with many advantages, such as unique fluorescence properties, anti-photobleaching, low toxicity, fine biocompatibility and high penetration depth in tissues, have been considered as a potential candidate in cell imaging fluorescent probes. Herein, N, S-codoped green-emissive CQDs (QY= 64.03%) were synthesized by the one-step hydrothermal method, with m-phenylenediamine as the carbon and nitrogen source, and L-cysteine as the nitrogen and sulfur dopant, under the optimum condition of 200 °C reaction for 2 h. Their luminescence was found to originate from the surface state. In light of the satisfactory photobleaching resistance and the low cytotoxicity, CQDs were used as a cell imaging probe for HeLa cell imaging. The results clearly indicate that cells can be labeled with CQDs, which can not only enter the cytoplasm, but also enter the nucleus through the nuclear pore, showing their broad application prospect in the field of cell imaging.     

Locating inositol 1,4,5-trisphosphate in the nucleus and neuronal dendrites with genetically encoded fluorescent indicators

Inositol 1,4,5-trisphosphate (InsP3) is a key second messenger in many cell types and also in distinct subcellular regions of single living cells; however, little is examined about the subcellular dynamics of InsP3 in a variety of cell types. We have developed fluorescent indicators to locate InsP3 dynamics in single living cells based on an intramolecular fluorescence resonance energy transfer. Our indicator has visualized InsP3 dynamics in the cytoplasm of cultured cells and even in single thin dendrites of hippocampal neurons, which has been unseen previously. We have further localized the present indicator in the nucleus and pinpointed nuclear InsP3 dynamics. The observation with our nuclear InsP3 indicator has solved a question on nuclear propagation of InsP3 from the cytoplasm and has drawn a conclusion that the nuclear InsP3 dynamics synchronously occurs with cytosolic InsP3 dynamics evoked by agonist stimulations. The present approach contributes to the understanding of when, where, and how InsP3 is generated and removed in a variety of living cells.