杉木生殖生物学特性的研究

本文就杉木大小孢子、雌雄配子体发育、受精作用、新细胞质形成以及细胞质遗传进行较为详细的研究，主要结果如下： 1.2月底小孢子母细胞进入减数分裂，其核相的变化与通常所描述的一致，细胞器集中排列在赤道板附近，其中质体有一重建的过程。当分裂完成时，在四个核之间产生胼胝质壁，细胞器随机分配在四分体中，每个四分孢子含有线粒体、质体、核糖体、高尔基体、脂体、RER和一些液泡。 2.花粉外壁的形成开始于四分体。颗粒状外壁外层和纤维状的原外壁几乎同时产生，具有三层结构的片层状外壁内层的形成晚于外壁外层。初期外壁外层的物质由小孢子和绒毡层共同提供，后期则主要由绒毡层形成的乌氏体所叠加;外壁内层则由小孢子本身分泌的物质形成，在液泡化之前厚度及数目达到最大，片层之间存在广泛的交叉和融合。3月底花粉成熟，花粉壁由颗粒状外层、片层结构的外壁内层和厚的纤维状内壁所组成，在喙处只有一层外壁内层和内壁。各种细胞器的活动在四分体及游离小孢子时期最为活跃。 3.绒毡层的变化与小孢子的发育密切相关，前者具有发生、发展和解体的过程。期间绒毡层分泌的圆球体在结构上有所不同，减数分裂时为中间电子密度深、外围浅的圆球体结构;四分体及游离小孢子时为中间电子密度浅、外围沉积有孢粉素的乌氏体;成熟花粉时为星芒状的乌氏体，它们共同形成花粉的我壁。绒毡层细胞中的细胞器也经历一个变化过程，造粉体的含量在四分体时达到高潮，在小孢子液泡期时基本消耗殆尽并解体;大量的粗糙内质网成零分布在质膜处，并与质膜走向平行。绒毡层解体时出现特殊的现象，具多个由多层膜组成的吞噬泡，并成堆分布，RER和线粒体为最后解体的细胞器。绒毡层外切向面的周绒毡层膜由两层膜组成，其上分布有大量的孢粉素和乌氏体。 4.萌发的花粉管4月底穿过珠心顶端，内含两个不育核和一精原细胞，不育核分布在具有淀粉、脂滴、线粒体和RER的原花粉细胞质中，位于精原细胞之前，这种关系保持到受精前。6月初精原细胞体积迅速增加到最大，相应的细胞质也发生变化，以线粒体为主的细胞质中出现少量质体，线粒体再资分化，细胞质中含有大量的可溶性多糖。 5.受精前，精原细胞分裂产生两个大小和形状相同的精细胞，彼此之间由胞间连丝的横壁联系在一起，并被原花粉细胞质所包被。精细胞中质体和淀粉粒的数量大幅度增加。精核们于细胞的中央，细胞质呈现区域分布的特点，据所含细胞器的差别，人核膜向外，细胞质分为五层，中间两层最为突出，即线粒体层和淀粉层，其间有大量核糖体的存在，线粒体层位于淀粉层的里面。这两层占据精细胞的大部分体积。 6.雌配子体游离核时期持续时间较长，近两个月，而从细胞化到卵细胞成熟则非常迅速，大约只需两周左右的时间。进一步发育，颈卵器中的液泡减少，细胞质变浓。初期少量的质体和淀粉粒被膨大的内质网片段和小泡的融合而与细胞质相隔，并最终退化。小内含物增加，亲锇颗粒出现。 7.成熟的卵细胞中具有大量各种形式的小内含物，细胞质被平行和环形的内质网所分隔。核膜外围有一圈疏松排列的亲锇颗粒，核仁变为多个基本为圆形的小核仁。没有质体的存在，大量脱分化的线粒体和核糖体集中分布在卵核的下部。 8.受精作用主要发生在6.9-6.16日期间，雄性细胞质始终伴随着精核向卵核移动，当两核接近时，朝向精核－面的卵核形成凹陷内，性细胞质覆盖在精核之上并最终包围两性核，而把雌性细胞质排除在外。因此，受精卵周围的胞质主要为由质体、线粒体和核糖体组成的新细胞质，且线粒体和质体的分布形式与精细胞的相同，即线粒体在里层，质体在外层。 9.融合后的合子核随即进行有丝分裂，形成两个原胚游离核，两个游离核同步分裂，并向基部移动，游离核始终分布在新细胞质中。八游离核时形成细胞壁，原胚属标准型。胚细胞中基本不含淀粉粒，具有大量的线粒体、高尔基体、RER和原质体。原胚之上的卵细胞质退化解体。杉木的质体和线粒体均为父本遗传。 10.幼胚的胚性细胞和胚柄细胞具有明显的差别，主要表现在质体的存在形式和高尔基体的数量上。胚性细胞中，原质体分布在核周围，大量的RER、高尔基体和线粒体平行于胚的走向;位于其上的胚柄细胞则含有淀粉粒和特别多的高尔基体。具简单多胚和裂生多胚，胚柄系统发达，7月底出现根原始细胞，8月中旬胚分化完成，具有两个子叶。

水松生殖生物学的研究

本文运用组织化学技术、透射电镜技术，就水松雌雄配子体发育，精卵细胞形成和结构，受精作用以及细胞质遗传等问题进行了较为详细的研究。主要结果如下： 1 绒毡层发育与雄配子体发育，尤其与花粉外壁的形成关系密切。绒毡层组织在小孢子母细胞和减数分裂时期达到发育高峰。四分体阶段和游离小孢子发育前期，绒毡层细胞中内质网、脂体、线粒体非常活跃，参与孢粉素和乌氏体的形成和转移。乌氏体有球形、星芒状两种类型，它们主要参与花粉外壁内层和外壁外层的建成。小孢子发育后期绒毡层细胞开始明显解体。 2 减数分裂阶段，质体、线粒俺等细胞器和造粉体随细胞核和染色体的变化，出现有规律的迁移现象。减数分裂前期，细胞核移到细胞的一侧;各种细胞器和造粉体迁移到细胞的另一侧，并随染色体移向细胞中部而转移到细胞质周缘。减数分裂中期l和后期，各种细胞器和造粉体汇聚在赤道板丙侧分布。说明细胞质中微管系统在起调节作用。 3 花粉壁形成开始于四分体时期。片层状结构的外壁内层随纤维状原外壁的出现雨形成。外壁外层和花粉内壁在小孢子发育中期几乎同时形成。小孢子细胞和绒毡层组织共同参与了外壁内层和外壁外层孢粉素物质的合成、转运。水松成熟花粉由孢粉素组成的外壁外层、片层状结构的外壁内层及有分层状结构的内壁构成。 4 传粉到受精间隔要四个月左右的时间。六月上旬，花粉管和精原细胞抵达颈卵器上部。精原细胞富含淀粉粒、质体、线粒体和异形泡并分区分布。受精前，精原细胞分裂形成两个大小和形状相同豹精细胞。精细胞含有质体、线粒体和异型泡等细胞器。 5 雌配子体游离核持续时间长，从颈卵器原始细胞形成到卵细胞发育成熟所需时间较短。复合颈卵器结构，颈细胞和套层细胞形态结构特殊。中央细胞不经分裂，直接行使卵细胞的功能。 6 成熟卵细胞中除弋量各种形式的内含物外，细胞质被庞大的内质网包围、分割，形成网膜系统。质体、淀粉等细胞器被包含成大内含物。在卵核周围细胞质中没有发现质体和发育完好的线粒体存在。 7 受精作用发生在六月十日左右。雌雄核融合时，朝向精核一面的卵核形成凹陷，精核陷入其中与卵核进一步融合。精卵融合可发生在颈卵器的上部、中部、下部甚至底部。有旋转受精、两个精核同时与一个卵核受精及多卵细胞现象等。 8 新细胞质主要为雄性细胞质成分。合子转移到颈卵器基部分裂，形成原胚游离核。合子、新细胞质和游离核周围淀粉鞘显著。八游离核时期形成细胞壁，新细胞质和淀粉粒转移到原胚细胞中。 9 水松的质体和线粒体为父系遗传。

Vortex filament simulation of trailing vortex merger

Aircraft in high-lift configuration shed multiple vortices. These generally merge to form a downstream wake consisting of two counter-rotating vortices of equal strength. The understanding of the merger of two co-rotating trailing vortices is important in evaluating the separation criteria for different aircraft to prevent wake vortex hazards during landing and take-off. There is no existing theoretical method on the basis of which such norms can be set. The present study is aimed at gaining a better understanding of the behaviour of wake vortices behind the aircraft. Two dimensional studies are carried out using the vortex blob method and compared with Bertenyi's experiment. It is shown that inviscid two dimensional effects are insufficient to explain the observations. Three dimensional studies, using the vortex filament method, are applied to the same test case. Two Lamb-Oseen profile vortices of the same dimensions and initial separation as the experiment are allowed to evolve from a straight starting condition until a converged steady flow is achieved. The results obtained show good agreement with the experimental distance to merger. Core radius and separation behaviour is qualitatively similar to experiment, with the exception of rapid increases at first. This may be partially attributable to the choice of filament-element length, and recommended further work includes a convergence study for this parameter. Copyright © 2005 by the American Institute of Aeronautics and Astronautics, Inc. All rights reserved.

Development of cell culture from caudal fin and heart of Tor tor (Hamilton-Buchanan)

Tor mahseer (Tor tor), possess high commercial and recreational value as they are potential game as well as food fish of India. Two cell culture systems were developed from fin and heart of T. tor (Hamilton-Buchanan). The explants excised aseptically from fingerling of T. tor were cultured in Leibovitz-15 (L-15) medium with 20% fetal bovine serum (FBS). Radiation of cells started after 72 hours and 48 hours of explant attachment from caudal fin and heart respectively. Confluent monolayer of cells with heterogeneous morphology around fin explants was observed after 7-10 days, where as a homogenous confluent layer of fibroblastic cells from heart explant was observed after 12-13 days. The establishment of cell culture systems from different organs and tissues of commercial important species would facilitates in vitro research.

Serum-free culture of rhesus monkey embryonic stem cells

Previous studies have shown that the maintenance and proliferation of undifferentiated rhesus monkey embryonic stem (rES) cells requires medium supplemented with fetal bovine serum (FBS). Due to the uncharacterized composition and variation in serum nature, the present study aimed to replace the serum-containing medium with a serum-free medium in the rES cell culture. The results showed that after the initial 48-h culture in the routinely used serum-containing medium, rES cells can grow and proliferate for a prolonged period in the serum-free medium composed of DMEM supplemented with a cocktail of BSA, IGF-1, TGF-alpha, bFGF, aFGF, estradiol, and progesterone. rES cells cultured in the serum-free medium maintained high level of alkaline phosphatase activity and OCT4 level. There was no indication of differentiation as judged by the marker gene expression of all three embryonic germ layers and trophoblast. In addition, serum-free culture would not affect the passage capacity and differentiation potential of rES cells. This work will facilitate the future study of induced differentiation of rES cells and other applications.

胎牛血清促进色素上皮衍生因子在皮肤角质形成细胞和成纤维细胞中的表达

下载PDF阅读器目的:探讨表皮角质形成细胞和真皮成纤维细胞中色素上皮衍生因子(PEDF) 表达的影响因素.方法:体外培养角质形成细胞和成纤维细胞,并以10%FBS(胎牛血清)刺激,通过免疫荧光和Western blot检测PEDF的表达.结果:PEDF大多位于细胞浆中,但在细胞核中也有少量表达.细胞浆中PEDF并非均质型分布,而是呈细颗粒状集聚.10%FBS促进角质形成细胞和成纤维细胞中PEDF的集聚和表达,而且这一分布形式不受组胺和佛波肉豆蔻醋酸(PMA) 的影响.结论:10% FBS促进角质形成细胞和成纤维细胞中PEDF的集聚和表达.

人胚胎神经干细胞的分离培养及鉴定研究

目的:研究人胚胎神经干细胞的培养条件及体外分化情况。方法:从12周龄人胚胎脑皮质分离细胞,采用无血清培养技术,协同应用碱性成纤维细胞生长因子(bFGF)、表皮生长因子(EGF)和重组人白血病抑制因子(rhLIF)进行培养;5-溴脱氧尿嘧啶核苷(BrdU)标记检测细胞的增殖能力,间接免疫荧光化学法检测细胞的分化情况。结果:培养得到的大量半悬浮生长的神经干细胞球能够传代培养;BrdU标记阳性,可诱导分化为神经元、星形胶质细胞和少突胶质细胞。结论:人胚胎脑皮质分离细胞培养得到的细胞群具有神经干细胞的基本特征,可进一步用于基础及临床研究。

HORMONAL-REGULATION OF TISSUE-TYPE PLASMINOGEN-ACTIVATOR AND PLASMINOGEN-ACTIVATOR INHIBITOR TYPE-1 IN CULTURED MONKEY SERTOLI CELLS

Sertoli cells play a central role in the control and maintenance of spermatogenesis. Isolated Sertoli cells of mouse and rat testes have been shown to secrete plasminogen activator (PA) and a plasminogen activator inhibitor type-1 (PAI-1) in culture. In this study, we have investigated the hormonal regulation of PA and PAI-1 activities in cultured monkey Sertoli cells. Sertoli cells (5x10(5) cells/well) isolated from infant rhesus monkey testes were preincubated at 35 degrees C for 16 h in 24-well plates precoated with poly(D-lysine) (5 mu g/cm(2)) in 0.5 mi McCoy's 5a medium containing 5% of fetal calf serum and further incubated for 48 h in 0.5 mi serum-free medium with or without various hormones or other compounds, PA as well as PAI-1 activities in the conditioned media were assayed by fibrin overlay and reverse fibrin autography techniques respectively. The Sertoli cells in vitro secreted only tissue-type PA (tPA), no detectable amount of urokinase-type PA (uPA) could be observed, Monkey Sertoli cells were also capable of secreting PAI-1, Immunocytochemical studies indicated that both tPA and PAI-1 positive staining localized in the Sertoli cells, spermatids and residual bodies of the seminiferous epithelium; Northern blot analysis further confirmed the presence of both tPA and PAI-1 mRNA in monkey Sertoli cells. Addition of follicle-stimulating hormone (FSH) or cyclic adenosine monophosphate (cAMP) derivatives or cAMP-generating agents and gonadotrophin-releasing hormone (GnRH) agonist or phorbol ester (PMA) to the cell culture significantly increased tPA activity. PAI-1 activity in the culture was also enhanced by these reagents except 8-bromo-dibutyryl-cAMP, forskolin and 3-isobutyl-1-methylxanthin (MIX) which greatly stimulated tPA activity, whereas decreased PAI-1 activity, implying that neutralization of PAI-1 activity by tile high level of tPA in the conditioned media may occur. These data suggest that increased intracellular signals which activate protein kinase A (PKA), or protein kinase C (PKC) can modulate Sertoli cell tPA and PAI-1 activities, The concomitant induction of PA and PAI-1 by the same reagents in the Sertoli cells may reflect a finely tuned regulatory mechanism in which PAI-1 could limit the excession of the proteolysis.

Spontaneous emission from radiative chiral nematic liquid crystals at the photonic band-gap edge: An investigation into the role of the density of photon states near resonance

In this article, we investigate the spontaneous emission properties of radiating molecules embedded in a chiral nematic liquid crystal, under the assumption that the electronic transition frequency is close to the photonic edge mode of the structure, i.e., at resonance. We take into account the transition broadening and the decay of electromagnetic field modes supported by the so-called "mirrorless"cavity. We employ the Jaynes-Cummings Hamiltonian to describe the electron interaction with the electromagnetic field, focusing on the mode with the diffracting polarization in the chiral nematic layer. As known in these structures, the density of photon states, calculated via the Wigner method, has distinct peaks on either side of the photonic band gap, which manifests itself as a considerable modification of the emission spectrum. We demonstrate that, near resonance, there are notable differences between the behavior of the density of states and the spontaneous emission profile of these structures. In addition, we examine in some detail the case of the logarithmic peak exhibited in the density of states in two-dimensional photonic structures and obtain analytic relations for the Lamb shift and the broadening of the atomic transition in the emission spectrum. The dynamical behavior of the atom-field system is described by a system of two first-order differential equations, solved using the Green's-function method and the Fourier transform. The emission spectra are then calculated and compared with experimental data. © 2013 American Physical Society.

Spontaneous emission from radiative chiral nematic liquid crystals at the photonic band-gap edge: an investigation into the role of the density of photon states near resonance.

In this article, we investigate the spontaneous emission properties of radiating molecules embedded in a chiral nematic liquid crystal, under the assumption that the electronic transition frequency is close to the photonic edge mode of the structure, i.e., at resonance. We take into account the transition broadening and the decay of electromagnetic field modes supported by the so-called "mirrorless"cavity. We employ the Jaynes-Cummings Hamiltonian to describe the electron interaction with the electromagnetic field, focusing on the mode with the diffracting polarization in the chiral nematic layer. As known in these structures, the density of photon states, calculated via the Wigner method, has distinct peaks on either side of the photonic band gap, which manifests itself as a considerable modification of the emission spectrum. We demonstrate that, near resonance, there are notable differences between the behavior of the density of states and the spontaneous emission profile of these structures. In addition, we examine in some detail the case of the logarithmic peak exhibited in the density of states in two-dimensional photonic structures and obtain analytic relations for the Lamb shift and the broadening of the atomic transition in the emission spectrum. The dynamical behavior of the atom-field system is described by a system of two first-order differential equations, solved using the Green's-function method and the Fourier transform. The emission spectra are then calculated and compared with experimental data.

Establishment, characterization, and virus susceptibility of a new marine cell line from red spotted grouper (Epinephelus akaara)

A marine fish cell line from the snout of red spotted grouper Epinephelus akaara, a protogynous hermaphrodite, was established, characterized, and subcultured with more than 60 passages. The grouper snout cell line (GSC) cells multiplied well in Dulbecco's modified Eagle's medium (DMEM) medium supplemented with 10% fetal bovine serum. The optimal growth temperature was 25 degrees C, and morphologically the cells were fibroblastic. Chromosome analysis revealed that the GSC cell line has a normal diploid karyotype with 2n = 8st + 40t. A virus titration study indicated that the cells were susceptible to turbot Scophthalmus Maximus rhabdovirus (SMRV) (10(8.5) TCID50 ml(-1)), while the viral titer of frog Rana grylio virus 9807 (RGV(9807)) reached 10(3.5) TCID50 ml-1. The infection was confirmed by cytopathic effect (CPE), immunofluorescence, and electron microscopy experiments, which detected the viral particles in the cytoplasm of virus-infected cells, respectively. Further, significant fluorescent signals were observed when the GSC cells were transfected with pEGFP vector DNA, indicating their potential utility for transgenic and genetic manipulation studies.

Total and subcellular distribution of trace elements in the liver of a mother-fetus pair of Dall's porpoises (Phocoenoides dalli)

Total and subcellular hepatic Zn, Cu, Se, Mn, V, Hg, Cd, and Ag were determined in a mother-fetus pair of Dall's porpoises (Phocoenoides dalli). Except for higher fetal Cu concentration, all maternal elements were higher. Elements existed mostly in the cytosol of both animals except in the case of maternal Ag in the microsome and fetal Cu and Ag in the nuclei and mitochondria. In the maternal cytosol, Zn, Mn, Hg, and Ag were present in the high-molecular-weight substances (HMW); Se and V were present in the low-molecular-weight substances (LMW); Cu and Cd were mostly sequestered by metallothionein (MT). In the fetal cytosol, Zn, Se, Mn, Hg, Cd, and Ag were present in the HMW and V in the LMW, while Cu and Ag were mostly associated with MT. MT isoforms were characterized using the HPLC/ICP-MS. Two and four obvious peaks appeared in the maternal and fetal MT fractions, respectively. The highest elemental ion intensities were at a retention time of 7.8 min for the mother, and for the fetus the peak elemental ion intensities occurred at a retention time of 4.3 min, suggesting that different MT isoforms may be involved in elemental accumulation in maternal and fetal hepatocytosols. (C) 2003 Elsevier Ltd. All rights reserved.

Factors affecting the efficiency of somatic cell nuclear transplantation in the fish embryo

Procedures to improve somatic cell nuclear transplantation in fish were evaluated. We reported effects of nonirradiated recipient eggs, inactivated recipient eggs, different combinations between recipient eggs and donor cells, duration of serum starvation, generation number, and passage number of donor cells on developmental rates of nuclear transplant (NT) embryos. Exposure to 25,000 R of gamma-rays inactivated recipient eggs. Single nucleus of cultured, synchronized somatic cell from gynogenetic bighead carp (Aristichthys nobilis) was transplanted into nonirradiated or genetically inactivated unfertilized egg of gibel carp (Carassius auratus gibelio). There was no significant difference in developmental rate between nonirradiated and inactivated recipient eggs (27.27% vs. 25.71%, respectively). Chromosome count showed that 70.59% of NT embryos contained 48 chromosomes. It showed that most NT embryos came from donor nuclei of bighead carp, which was supported by microsatellite analysis of NT embryos. But 23.53% of NT embryos contained more than 48 chromosomes. It was presumed that those superfluous chromosomes came from nonirradiated recipient eggs. Besides, 5.88% of NT embryos were chimeras. Eggs of blunt-snout bream (Megalobrama amblycephala) and gibel carp were better recipient eggs than those of loach (Misgurnus anguillicaudatus) (25% and 18.03% vs. 8.43%). Among different duration of serum starvation, developmental rate of NT embryos from somatic nuclei of three-day serum starvation was the highest, reaching 25.71% compared to 14.14% (control), 20% (five-day), and 21.95% (seven-day). Cultured donor cells of less passage facilitated reprogramming of NT embryos than those of more passage. Recloning might improve the developmental rate of NT embryos from the differentiated donor nuclei. Developmental rate of fourth generation was the highest (54.83%) and the lowest for first generation (14.14%) compared to second generation (38.96%) and third generation (53.01%). (C) 2002 Wiley-Liss, Inc.

Studies on polysaccharides from three edible species of Nostoc (Cyanobacteria) with different colony morphologies: Comparison of monosaccharide compositions and viscosities of polysaccharides from field colonies and suspension cultures

Hot water-soluble polysaccharides woe extracted from field colonies and suspension cultures of Nostoc commune Vaucher, Nostoc flagelliforme Berkeley et Curtis, and Nostoc sphaeroides Kutzing. Excreted extracellular polymeric substances (EPS) were isolated from the media in which the suspension cultures were grown. The main monosaccharides of the field colony polysaccharides from the three species were glucose, xylose, and galactose, with an approximate ratio of 2:1:1. Mannose was also present, but the levels varied among the species, and arabinose appeared only in N. flagelliforme. The compositions of the cellular polysaccharides and EPS from suspension cultures were more complicated than those of the field samples and varied among the different species. The polysaccharides from the cultures of N. flagelliforme had a relatively simple composition consisting of mannose, galactose, glucose, and glucuronic acid, but no xylose, as was found in the field colony polysaccharides. The polysaccharides from cultures of N. sphaeroides contained glucose (the major component), rhamnose, fucose, xylose, mannose, and galactose. These same sugars were present in the polysaccharides from cultures of N. commune, with xylose as the major component. Combined nitrogen in the media had no qualitative influence on the compositions of the cellular polysaccharides but affected those of the EPS of N. commune and N. flagelliforme. The EPS of N. sphaeroides had a very low fetal carbohydrate content and thus was not considered to be polysaccharide in nature. The field colony polysaccharides could be separated by anion exchange chromatography into neutral and acidic fractions having similar sugar compositions. Preliminary linkage analysis showed that 1) xylose, glucose, and galactose were 1-->4 linked, 2) mannose, galactose, and xylose occurred as terminal residues, and 3) branch points occurred in glucose as 1-->3,4 and 1-->3,6 linkages and in xylose as a 1-->3,4 linkage. The polymer preparations from field colonies had higher kinematic viscosities than those from corresponding suspension cultures. The high viscosities of the polymers suggested that they might DE suitable for industrial uses.