用共聚焦扫描显微镜检术和酶法分离技术观测观察水稻胚囊发育

本文中以水稻为实验材料，用共聚焦扫描显微镜检术(CLSM)和酶法分离技术观察其胚囊取得好的效果，主要实验结果如下： 1.水稻子房分别用FAA和4%戊二醛固定液固定，经乙醇脱水，然后用冬青油整体透明及丁香油封片。在共聚焦扫描显微镜下，发育中的水稻胚囊显示自发荧光，可分辨出胚囊内部结构。FAA和4%戊二醛两种固定液效果不同，后者效果更佳。用上述实验程序进一步观察拟南芥菜胚囊及水稻和拟南芥菜花粉表明，CLSM可用于快速观察分析水稻和拟南芥菜胚囊发育过程以及水稻胚囊败育情况，但不适用于观察分析其花粉发育过程。 2.水稻子房用FAA、FPA或卡诺氏固定液固定，经乙醇转至蒸镏水，然后置于由纤维素酶和果胶酶组成的酶液中，在37℃保温条件下进行连续振荡酶解，在振荡过程中辅以手工显微操作，由此即可分离出水稻固定胚珠的胚囊。用相差显微镜观察分离的胚囊表明，胚囊整体性及立体感强，观察效果好。在成功分离水稻固定材料胚囊的基础上，初步尝试了水稻新鲜材料胚囊的分离。

松杉类植物花粉发育和传粉生物学的研究

本文用光学显微镜结合荧光技术对青扦花粉的发育过程进行了观察;用共聚焦显微镜观察了白扦生长花粉管细胞内的游离Ca2+分布;利用原子力显微镜对雪松和水杉花粉外壁的亚结构进行了研究：用透射电镜、扫描电镜及解剖镜等技术研究了侧柏、北美香柏、红豆杉、粗榧和白皮松的传粉机制，结果如下。 青扦花粉的发育过程与松科其它一些植物花粉的发育模式相似。从小孢子母细胞到成熟花粉约二十天左右。小孢子母细胞进入减数分裂前彼此分开，但在某些部位仍有连接。细胞质内有大量淀粉粒，在减数分裂过程中减少或消失，没有观察到明显的淀粉粒带。减数分裂中的胞质分裂为同时型，四分体为四面体型。小孢子刚从四分体释放出来时，气囊已开始形成，细胞中含大量淀粉粒。随着小孢子的发育，其体积增大，并出现液泡，细胞核移向一侧。小孢子第一次不对称分裂产生一个大的中央细胞和一个小的原叶细胞。中央细胞不久就进行第二次分裂产生精子器原始细胞和第二原叶细胞。原叶细胞形成后，其与中央细胞或精子器原始细胞之间的壁逐渐沉积胼胝质，以后随着原叶细胞的退化，胼胝质壁消失。精子器原始细胞分裂形成管细胞和生殖细胞，生殖细胞在散粉前分裂形成体细胞（精原细胞）和柄细胞（不育细胞）。成熟花粉为5细胞，但两个原叶细胞已退化消失。 白扦花粉在10%蔗糖+0.01%硼酸的液体培养基内培养12小时后开始萌发。在正常生长的花粉管中，其顶端有一个透明区，而透明区后则含有大量的贮藏物质颗粒。在停止生长的花粉管中透明区消失，而整个花粉管顶端也被储藏物质颗粒充满。正常生长的花粉管顶端有一个较高的Ca2+浓度。在停止生长的花粉管内不具有这样一个Ca2+梯度。 雪松和水杉二种花粉外壁中由孢粉素构成的亚结构单位形态相似，均呈颗粒状，但大小略有不同。雪松的长56-99 nm，宽42-74;水杉的长81-118 nm，宽43-98 nm。在雪松中这些亚单位紧密排列组成短棒状或球状的花粉外壁结构单位，再由几个到十几个这样的结构单位组成较大的岛屿状结构。在这些岛屿状结构之间有大小不一的空隙存在，整个花粉外壁由这样一些岛屿状结构交互连接形成。水杉花粉外壁的亚单位排列也较紧密，且有3-10个成群分布的趋势，但各群之间界限不明显。此外，雪松和水杉的花粉外壁亚单位均无螺旋状排列趋势，这一结果倾向于支持Southworth关于花粉外壁亚单位颗粒状并呈网状排列的观点。 白皮松胚珠倒生，其发育过程与松属的其它种相似，成熟胚珠珠孔端具两手臂状结构，有利于接收花粉。花粉具气囊。传粉期间，没有观察到传粉滴产生，但珠心顶端细胞解体形成花粉室。花粉室内可接受一至几个花粉，花粉在花粉室内的位置无明方向性。传粉时，胚珠处于大孢子线细胞时期。花粉在花粉室内萌发形成花粉管进入珠心组织，花粉管在珠心内生长一段时间后停止生长，并于次年春天重新启动生长。离体生长的花粉管顶端常有胼胝质产生，但顶端区域后的花粉管壁上却无胼胝质沉积。 侧柏、北美香柏、红豆杉和粗榧均为直生胚珠。传粉时胚珠产生传粉滴。在红豆杉胚珠发育早期，珠心表面细胞轮廓清晰;而在后期，其珠心表面则形成了一层膜状结构。这层膜状结构在传粉前随珠心细胞的解体而破裂，珠心细胞的降解产物参与了传粉滴形成。在传粉前和传粉期，珠心细胞内含大量的线粒体、内质网、高尔基体和小泡。传粉滴主要由珠心细胞分泌形成。这四种植物的花粉均无气囊，属可湿性花粉。红豆杉和粗榧的花粉水合时，内壁膨胀，外壁开裂。通常情况下，红豆杉花粉的外壁保留在传粉滴的表面，而花粉的其它部分沉入传粉滴内。侧柏和北美香柏的传粉滴授粉后，花粉进入传粉滴导致传粉滴的明显收缩。在侧柏中传粉滴授粉后100分钟内就完全收缩进入珠孔。传粉滴收缩的速率与所授花粉数量和花粉的种类有关。与侧柏亲缘关系较近植物花粉引起传粉滴的收缩速率和侧柏自身花粉引起的传粉滴收缩速率相似;反之，收缩速率变慢。侧柏传粉滴的收缩可能主要是由于花粉减弱胚珠分泌的结果。但授粉不引起红豆杉和粗榧传粉滴的明显收缩。在红豆杉和粗榧中，从授粉到传粉完全收缩需要20-24小时。这两种植物传粉滴的收缩可能主要是蒸发引起的非代谢性过程，与侧柏和美香柏属于不同的传粉滴收缩机制。

Genome-wide SNP and haplotype analyses reveal a rich history underlying dog domestication

Advances in genome technology have facilitated a new understanding of the historical and genetic processes crucial to rapid phenotypic evolution under domestication(1,2). To understand the process of dog diversification better, we conducted an extensive genome-wide survey of more than 48,000 single nucleotide polymorphisms in dogs and their wild progenitor, the grey wolf. Here we show that dog breeds share a higher proportion of multi-locus haplotypes unique to grey wolves from the Middle East, indicating that they are a dominant source of genetic diversity for dogs rather than wolves from east Asia, as suggested by mitochondrial DNA sequence data(3). Furthermore, we find a surprising correspondence between genetic and phenotypic/functional breed groupings but there are exceptions that suggest phenotypic diversification depended in part on the repeated crossing of individuals with novel phenotypes. Our results show that Middle Eastern wolves were a critical source of genome diversity, although interbreeding with local wolf populations clearly occurred elsewhere in the early history of specific lineages. More recently, the evolution of modern dog breeds seems to have been an iterative process that drew on a limited genetic toolkit to create remarkable phenotypic diversity.

Relationship between trichosanthin cytotoxicity and its intracellular concentration

Trichosanthin (TCS) is a type I ribosome-inactivating protein with board spectrum of biological activity. Toxicity of this compound differs in different cell lines and this study examined the cause of such difference. It is generally believed that TCS toxicity is mediated through intracellular ribosome inactivation. Therefore, TCS toxicity should be determined by the amount inside cells rather than outside. Three different cell types IC21, JAR and Vero cell lines were chosen with high, medium and low sensitivity to TCS. Intracellular concentrations of fluorescein isothiocyanate labeled TCS were determined by laser scanning confocal microscopy. A good relationship was demonstrated between intracellular TCS concentration and toxicity. Highest intracellular concentration was found in IC21, followed by JAR, and lowest in Vero cells. When the intracellular TCS concentrations in these cells were reduced by using a competitive inhibitor to block cell entry, cytotoxicity was not observed. In conclusion, there is strong evidence to indicate that cytotoxicity of TCS is dependent on its intracellular concentration. Variation of cytotoxicity in different cells may be related to the mechanisms affecting its internalization. (C) 2002 Published by Elsevier Science Ireland Ltd.

口腔移植材料与成骨细胞相容性的激光共聚焦显微镜观察

下载PDF阅读器目的:观察不同处理的钛金属表面与成骨细胞的生物相容性.方法:利用共聚焦显微镜荧光信号通过物镜返回光电倍增管成像的原理,获取不透光的钛金属表面图像,对钛金属表面(打磨、喷砂、喷砂酸蚀表面)接种的成骨细胞骨架进行荧光标记,并用共聚焦显微镜获取荧光图像,观察细胞和钛金属表面的生物相容性,并且通过逐渐深入的多层扫描,探索细胞和移植材料结合的进一步信息.结果:喷砂表面适合成骨细胞的贴附和生长.结论: 钛金属与成骨细胞的结合情况主要与金属表面的物理形态有关.

Localization and distribution of tissue type and urokinase type plasminogen activators and their inhibitors type 1 and 2 in human and rhesus monkey fetal membranes

Fetal membranes consist of 10 distinct layers including components of amnion, chorion and decidua, the latter being of maternal origin. They form mechanically integrated sheets capable of retaining amniotic fluid and play an essential role in protecting fetal growth and development in the pregnant uterus. The extracellular matrix, substrate for plasminogen activators (PAs), is an important supportive framework of the fetal membranes. :Fetal membranes from women with preterm premature rupture of membranes may differ in their protease activity compared with normal membranes. To identify the presence of PAs and their inhibitors (PAI) and their possible role in the process of fetal membrane rupture, this study in investigated the distribution and localization of both protein and mRNA for tissue (t) and urokinase (u) PA and their inhibitors type 1 (PAI-1) and type 2 (PAI-2) in amniochorion of human and rhesus monkey using conventional and. confocal immunofluorescence microscopy. In situ hybridization analysis showed that the distribution and localization of mRNAs for tPA, uPA, PAI-I and PAI-2 were similar in the fetal membranes of human and rhesus monkey; no obvious species difference was observed. Evidence of tPA mRNA was detected in amniotic epithelium, trophoblast cells and nearly all cells of the decidual layer. Strong expression of uPA mRNA was noted in the decidual cells which increased in intensity as the abscission point was approached. Weak staining in chorion laeve trophoblast was also detected. In situ hybridization experiments showed PAI-1 mRNA to be concentrated mainly in the decidual cells, some of which were interposed into the maternal-facing edge of the chorion laeve. Maximal labelling of the decidua occurred towards the zone of abscission. Weak expression of PAI-1 mRNA nas also noted in some cells of the chorion laeve. The distribution of PAI-2 mRNA in amniochorion was also concentrated in the cells of the decidual layer, maximum expression of the mRNA was in the level of abscission. No detectable amount of mRNAs for tPA, uPA, PAI-1 and PAI-2 was found in the fibroblast, reticular and spongy layers. Distribution of the proteins of tPA, uPA and PAI-1 in the fetal membranes of these two species was consistent with the distribution of their mRNA. Anti-PAI-2 immunofluorescence was found to be strongly concentrated in the amniotic epithelium, but PAI-2 mRNA was negative in this layer, suggesting that the epithelium-associated PAI-2 is not of epithelial origin. These findings suggest that a local fibrinolysis in fetal membranes generated by precisely balanced expression of PAs and their inhibitors via paracrine or autocrine mechanisms may play an essential role in fetal membrane development, maturation and in membrane rupture. Following an analysis of the distribution and synthesis of activators and inhibitors it was found that they may play a role in abscission during the third stage of labour. (C) 1998 W. B. Saunders Company Ltd.

Cell surface display of functionally active lipases from Yarrowia lipolytica in Pichia pastoris

The lipase genes of Yarrowia lipolytica, LIPY7 and LIPY8, fused with FLO-flocculation domain sequence from Saccharomyces cerevisiae at their N-termini, were expressed in Pichia pastoris KM71. Following the induction with methanol, the recombinant proteins were displayed on the cell surface of P. pastoris, as confirmed by the confocal laser scanning microscopy. The LipY7p and LipY8p were anchored on P. pastoris via the flocculation functional domain of Flo 1 p. The surface-displayed lipases were characterized for their application as the whole-cell biocatalyst. These lipases can also be cleaved off from their anchor by enterokinase treatment to yield functionally active proteins in the supernatant offering an alternative purification method for LipY7p and LipY8p. (c) 2007 Elsevier Inc. All rights reserved.

Characterization of the Rana grylio virus 3 beta-hydroxysteroid dehydrogenase and its novel role in suppressing virus-induced cytopathic effect

The 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) isoenzymes play a key role in cellular steroid hormone synthesis. Here, a 3 beta-HSD gene homolog,was cloned from Rana grylio virus (RGV), a member of family Iridoviridae. RGV 3 beta-HSD gene has 1068 bp, encoding a 355 aa predicted protein. Transcription analyses showed that RGV 3 beta-HSD gene was transcribed immediate-early during infection from an initiation site 19 nucleotides upstream of the translation start site. Confocal microscopy revealed that the 3 beta-HSD-EGFP fusion protein was exclusively colocalized with the mitochondria marker (pDsRed2-Mito) in EPC cells. Upon morphological observation and MTT assay, it was revealed that overexpression of RGV 3 beta-HSD in EPC cells could apparently suppress RGV-induced cytopathic effect (CPE). The present studies indicate that the RGV immediate-early 3 beta-HSD gene encodes a mitochondria-localized protein, which has a novel role in suppressing virus-induced CPE. All these suggest that RGV 3 beta-HSD might be a protein involved in host-virus interaction. @ 2006 Elsevier Inc. All rights reserved.

Stress and its effect on optical properties of GaN epilayers grown on Si(111), 6H-SiC(0001), and c-plane sapphire

The stress states in unintentionally doped GaN epilayers grown on Si(111), 6H-SiC(0001), and c-plane sapphire, and their effects on optical properties of GaN films were investigated by means of room-temperature confocal micro-Raman scattering and photoluminescence techniques. Relatively large tensile stress exists in GaN epilayers grown on Si and 6H-SiC while a small compressive stress appears in the film grown on sapphire. The latter indicates effective strain relaxation in the GaN buffer layer inserted in the GaN/sapphire sample, while the 50-nm-thick AlN buffer adopted in the GaN/Si sample remains highly strained. The analysis shows that the thermal mismatch between the epilayers and the substrates plays a major role in determining the residual strain in the films. Finally, a linear coefficient of 21.1+/-3.2 meV/GPa characterizing the relationship between the luminescent bandgap and the biaxial stress of the GaN films is obtained. (C) 2003 American Institute of Physics.

Establishment and characterization of dual-species biofilms formed between a 3,5-dinitrobenzoic-degrading strain and bacteria with high biofilm-forming capabilities

In this study, the possibility of establishing a dual-species biofilm from a bacterium with a high biofilm-forming capability and a 3,5-dinitrobenzoic acid (3,5-DNBA)-degrading bacterium, Comamonas testosteroni A3, was investigated. Our results showed that the combinations of strain A3 with each of five strains with a high biofilm-forming capability (Pseudomonas sp. M8, Pseudomonas putida M9, Bacillus cereus M19, Pseudomonas plecoglossicida M21 and Aeromonas hydrophila M22) presented different levels of enhancement regarding biofilm-forming capability. Among these culture combinations, the 24-h dual-species biofilms established by C. testosteroni A3 with P. putida M9 and A. hydrophila M22 showed the strongest resistance to 3,5-DNBA shock loading, as demonstrated by six successive replacements with DMM2 synthetic wastewater. The degradation rates of 3,5-DNBA by these two culture combinations reached 63.3-91.6% and 70.7-89.4%, respectively, within 6 h of every replacement. Using the gfp-tagged strain M22 and confocal laser scanning microscopy, the immobilization of A3 cells in the dual-species biofilm was confirmed. We thus demonstrated that, during wastewater treatment processes, it is possible to immobilize degrader bacteria with bacteria with a high biofilm-forming capability and to enable them to develop into the mixed microbial flora. This may be a simple and economical method that represents a novel strategy for effective bioaugmentation.

OPTICAL CHARACTERISTICS OF GAAS/ALGAAS RIDGE-QUANTUM-WELL-WIRES GROWN BY MBE ON NONPLANAR SUBSTRATES

With conventional photolithography and wet chemical etching, we have realized GaAs/AlGaAs buried ridge-quantum-well-wires (RQWWs) with vertically stacked wires in lateral arrays promising for device application, which were grown in situ by a single-step molecular beam epitaxy growth and formed at the ridge tops of mesas on nonplanar substrates. Confocal photoluminescence (CPL) and polarization-dependent photoreflectance (PR) are applied to study optical characteristics of RQWWs. Lateral bandgap modulation due to lateral variation of QW layer thickness is demonstrated not only by CPL but also by PR. As one evidence for RQWWs, a large blue shift is observed at the energy level positions for electronic transitions corresponding to quantum wells (QWs) at the ridge tops of mesas compared with those corresponding to QWs on nonpatterned areas of the same sample. The blue shift is in contradiction with the fact that the GaAs QW layers at the tops of the mesas are thicker than those on nonpatterned areas. The other evidence for RQWWs, optical anisotropy is provided by the polarization-dependent PR, which results from lateral quantum size effect existing at the tops of the mesas.

Detection of Copper Ion with Laser-Induced Fluorescence in A Capillary Electrophoresis Microchip

A capillary electrophoresis microchip coupled with a confocal laser-induced fluorescence (LIF) detector was successfully constructed for the analysis of trace amounts of heavy metals in environmental sources. A new fluorescence dye, RBPhOH, synthesized from rhodamine B, was utilized in a glass microchip to selectively determine copper with high sensitivity. A series of factors including running buffer concentration, detection voltage, and sample loading time were optimized for maximum LIF detector response and, hence, method sensitivity.

A new potential radiosensitizer- multi-walled carbon nanotubes modified by ammonium persulfate

Here we prepare carbon nanotubes modified with ammonium persulfate, very short carbon nanotubes with 50-100 nanometer length was obtained, and the higher P potential of 52 mV was detected, these supporting the successful modification. HeLa cells were irradiated with P rays via adding or absent above functionalized carbon nanotubes (f- WCNTs) into cell culture medium with different concentration and radiation dosage. Confocal microscopy images and fluorescence-labeled DNA detection verified the successfully pure multi-walled carbon nanotubes (p-WCNTs) and f-WCNTs penetrated into cells. Compared with pure radiation, by MTT test, f-WCNTs induced cell death markedly with about 8.7 times higher than former one under little dose of radiation; meanwhile, no obvious toxicity was observed both in p-WCNTs and f-WCNTs without of radiation exposure. We hypothesized that large amount of hydroxyl and carbonyl organs on the surface of very short f-WCNTs changed into free radicals result from radiations led cell damage. These implied that f-WCNTs could be regarded as a new radiosensitizer.

Raman spectroscopy of apatite irradiated with swift heavy ions with and without simultaneous exertion of high pressure

Durango apatite was irradiated with energetic U ions of 2.64 GeV and Kr ions of 2.1 GeV, with and without simultaneous exposure to a pressure of 10.5 GPa. Analysis by confocal Raman spectroscopy gives evidence of vibrational changes being marginal for fluences below 5x10(11) ions/cm(2) but becoming dominant when increasing the fluence to 8x10(12) ions/cm(2). Samples irradiated with U ions experience severe strain resulting in crystal cracking and finally breakage at high fluences. These radiation effects are directly linked to the formation of amorphous tracks and the fraction of amorphized material increasing with fluence. Raman spectroscopy of pressurized irradiated samples shows small shifts of the band positions with decreasing pressure but without a significant change of the Gruneisen parameter. Compared to irradiations at ambient conditions, the Raman spectra of apatite irradiated at 10.5 GPa exhibit fewer modifications, suggesting a higher radiation stability of the lattice by the pressure applied.

Swelling behaviour of alginate-chitosan microcapsules prepared by external gelation or internal gelation technology

Swelling behaviour is one of the important properties for microcapsules made by hydrogels, which always affects the diffusion and release of drugs when the microcapsules are applied in drug delivery systems. In this paper, alginate-chitosan microcapsules were prepared by different technologies called external or internal gelation process respectively. With the volume swelling degree (S-w) as an index, the effect of properties of chitosan on the swelling behaviour of both microcapsules was investigated. It was demonstrated that the microcapsules with low molecular weight and high concentration of chitosan gave rise to low S-w. Considering the need of maintaining drug activity and drug loading, neutral pH and short gelation time were favorable. It was also noticed that S-w of internal gelation microcapsules was lower than that of external gelation microcapsules, which was interpreted by the structure analysis of internal or external gelation Ca-alginate beads with the aid of confocal laser scanning microscope. (C) 2004 Elsevier Ltd. All rights reserved.