车前科和苦苣苔科一些典型类群花对称性基因的进化发育生物学研究—兼论地黄属和崖白菜属的系统位置

在模式植物金鱼草中的花对称性分子发育与遗传学研究揭示出相关调控基因在花对称性形成过程中的功能和表达式样及其相互作用机制，但被子植物中花对称性的繁杂多样远非模式植物的表达模式所能概括。因此，我们选择车前科和苦苣苔科中与金鱼草较近缘的典型类群地黄属和非洲紫罗兰属作为研究对象，针对它们在花对称性形成方面区别于金鱼草的不同式样，开展这些类群中花对称性主控基因CYCLOIDEA(CYC)类基因的进化发育生物学研究。该研究旨在探讨CYC 类基因的功能和表达式样的变化在进化上的内在联系。 地黄花对称性基因的进化发育研究结果显示，地黄中CYC 类基因RgCYC 的表达模式与CYC 基因在金鱼草中和McCYC 基因在Mohavea 中的表达模式存在明显的差异。首先， RgCYC 基因在近轴雄蕊预期发生位置表皮细胞下的强烈表达与地黄近轴雄蕊的缺失密切相关。转录因子中的氨基酸替代所导致蛋白质功能的改变使RgCYC 基因对细胞周期基因cyclin D3b 抑制作用的增强可能是地黄中近轴雄蕊原基发生过程被彻底阻断的主要原因。由此看来，CYC 类基因的作用不仅导致近轴花器官生长缓慢或退化，而且可能与自然类群中花近轴器官丢失的现象有关。其次，同McCYC 基因在Mohavea 中的表达模式相似，RgCYC 基因的表达也从近轴雄蕊延伸到了两侧雄蕊，但是并没有强烈地抑制两侧雄蕊的发育，仅仅使得两侧雄蕊短于远轴雄蕊从而在地黄中形成二强雄蕊。这一现象可能是由于RgCYC 基因的表达与McCYC 基因的表达在时间和空间上的差异所造成的，并显示地黄中二强雄蕊的形成机制和金鱼草完全不同。第三，RgCYC 基因在近轴花冠裂片的表达没有象CYC 在金鱼草中一样明显促进它们的生长。此外，近轴花冠裂片明显的自身对称性显示在地黄中RgCYC 基因在两侧对称性形成方面可能单独对近轴花器官进行调控。地黄中RgCYC 基因的表达模式反映了广义唇形目中从五数花到四数花进化过程的一种新的进化机制。 两侧对称花向次生辐射对称花的反演进化机制在花对称性进化发育研究中倍受关注。我们在苦苣苔科中选择非洲紫罗兰栽培品种作为研究材料，通过 mTAIL-PCR 分别在两侧对称花和辐射对称花的栽培品种中分离出了包含完整的 ORF 的CYC 类基因：SiCYC1A 和SiCYC1B。这两个基因的完整序列在DNA 水平的相似性为88%，均包含了完整的TCP domain, R domain 和 5’ 端区段。令人意外的是SiCYC1A 和SiCYC1B 这两个基因的DNA 序列在两侧和辐射对称花品种中均完全一致。根据对导致辐射对称花产生机制的比较分析，我们认为在这两个栽培品种中的SiCYC1A 和 SiCYC1B 基因可能存在着某一共同的调节因子对其进行调控。其可能途径是该调节因子同时调控SiCYC1A 和 SiCYC1B 基因，这一共同的调节因子的改变导致了SiCYC1A 和 SiCYC1B 基因部分或完全失去功能，从而使两侧对称花转变为辐射对称花。 崖白菜属的花部器官发生研究显示其花萼和花冠裂片的发生顺序与毛地黄族和婆婆纳族相似，花冠裂片早期生长的迟滞和花冠裂片折叠式样介于毛地黄族和婆婆纳族之间。但是，近轴雄蕊的发育缺失完全不同于毛地黄族中的其它类群。对地黄属和崖白菜属以及它们近缘类群的ITS 或trnL-F 序列所构建的系统树的分析显示，地黄属和崖白菜属呈姊妹群。然而，分子系统学研究结果并不支持传统系统学和个体发育研究对这两个属科级系统位置的认识。毛地黄属与婆婆纳属和车前属构成一个单系分支，而地黄属与崖白菜属则形成另外一个独立的分支，并与泡桐属与透骨草科所形成的分支首先聚在一支。因此，毛地黄族可能并不是一个单系类群，地黄属和崖白菜属的科级系统位置可能需要重新考虑。

大叶石上莲（苦苣苔科，马铃苣苔属）花对称性基因的表达模式和进化研究

模式植物金鱼草的花对称性分子发育及遗传学研究揭示了花对称性的相关调控基因在花发育过程种的表达模式和功能以及它们的相互作用机制。然而，被子植物的花对称性丰富多样，远非模式植物的模式所能概括。根据目前的研究我们选择苦苣苔科马铃苣苔属作为研究对象，选取其中具有代表性的种大叶石上莲开展控制花对称性的几个关键基因的进化发育生物学研究，初步探讨这些基因的表达模式和功能以及它们与花对称性进化的联系。 RT-PCR实验结果显示，在大叶石上莲中花对称性基因ObCYC, ObRAD, ObDIV与它们的同源基因CYC, RAD, DIV在金鱼草中的表达模式有着明显的差异，这也意味着这些基因的功能及相互作用也有所不同。首先，大叶石上莲的ObCYC1C基因的表达与金鱼草的CYC基因相似，结合花形态比较认为ObCYC1C基因在花器官近轴区域的表达抑制了背部花器官的发育，在构建大叶石上莲微弱的两侧对称花过程中起到了主导的作用; 其它的几个ObCYC基因有着完全不同于CYC的表达模式，ObCYC1D的表达延伸到了花腹部区域而ObCYC2A在花发育中期和晚期没有表达，这意味着它们可能具有除控制花对称性之外的功能; 另一个特殊的现象是ObCYC2B因为在可读框架中一个终止子的提前出现而失去了功能。其次，ObRAD, ObDIV的表达与RAD, DIV的表达差异也极为明显，通过系统树和氨基酸序列比对分析发现ObRAD, ObDIV与RAD, DIV的保守功能区序列存在明显的不同，而ObRAD, ObDIV与BlRAD, BlDIV的保守区很相似，这与它们的花对称性以及基因的表达模式相对映，同时也说明了保守功能区具有极重要的作用。本研究显示了大叶石上莲中的花对称性基因的表达模式和功能与模式植物金鱼草存在显著的差异，在此基础上开展更深入的进化发育生物学研究将有助于揭示花对称性基因的分子进化及其与花对称性表型进化的关系。

玄参科毛泡桐花对称性基因的分离和表达研究

花两侧对称性的获得是由多次独立的进化事件而完成。在被子植物进化过程中，花两侧对称性在植物的适应辐射过程中有重要意义。在模式植物金鱼草中，CYCLOIDEA（CYC）是控制花对称性的关键基因。近年来，CYC 基因的同源基因已经在多个类群中被分离出来，并开展了CYC 类基因的分子进化及其与花对称性表型进化相关关系的研究。 泡桐属是模式植物金鱼草的一个近缘类群，我们从泡桐属的毛泡桐中分离出了CYC基因的两个同源基因，分别命名为PtCYC1 和PtCYC2 。组织特异性RT-PCR显示他们的表达模式发生了明显的分化。 毛泡桐中PtCYC1 基因的表达模式与 CYC 基因在金鱼草中和 McCYC 基因在M. confertiflora 中的表达模式存在明显的差异但是和RgCYC 在地黄（Rehmannia）中的表达模式十分相似。PtCYC1 基因在近轴区域的强烈表达可能与毛泡桐近轴雄蕊的缺失有关。 尽管PtCYC1和M. confertiflora 中的McCYC 基因均从近轴雄蕊延伸到了两侧雄蕊，但PtCYC1 基因的后期表达较弱。因此，毛泡桐中的两侧雄蕊的发育并没有像M. confertiflora 中的两侧雄蕊受到强烈的抑制而成为退化雄蕊，仅仅使得两侧雄蕊短于两个远轴雄蕊从而在毛泡桐中形成二强雄蕊。地黄属和泡桐属两侧对称花的显著特征是缺乏近轴雄蕊。PtCYC1 和RgCYC基因的表达模式的高度一致显示出近轴雄蕊的缺失可能和CYC类基因的强烈表达有关。十分有趣的是毛泡桐中PtCYC2 基因在各个区域均没有表达。对于PtCYC2 基因是否还有其他方面的功能还有待于进一步探讨。

烟叶唇柱苣苔（Chirita heterotricha）两侧对称花的进化发育途径

被子植物菊亚纲原始类群的花为辐射对称花，伴随着适应性进化和传粉者的专性化，两侧对称花随之出现并快速进化和多样化。与花对称性相关的基因首先在模式植物金鱼草中被分离出来，它们包括TCP 基因家族的两个基因Cycloidea（CYC）和Dichotama（DICH），MYB 基因家族的两个基因Radialis（RAD）和Divaricata（DIV）。模式植物金鱼草的分子发育与遗传学研究初步揭示出在花对称性形成过程中相关调控基因的功能、表达模式及其相互作用机制。研究表明，金鱼草花中CYC 和DICH 基因只在背部表达，控制背部属性。DIV基因早期在所有部位表达，但只影响腹部属性。CYC 和DICH 通过激活与DIV具有颉抗作用的RAD 基因在背部的表达来抑制DIV 基因在背部的作用从而构建了金鱼草的两侧对称花。但是，被子植物繁杂多样的花对称性远非一种模式植物所能概括。此外，被子植物花对称性的演化也是一个悬而未决的问题。要全面了解被子植物花对称性的起源、多样化及其背后的决定机制，有必要进一步研究不同类群中花对称性相关基因的功能变化和进化式样。 苦苣苔科（Gesneriaceae）与金鱼草所属的车前科（Plantaginaceae）同属于菊亚纲的广义唇形目。研究表明广义唇形目的祖先已经具有了两侧对称花，苦苣苔科是广义唇形目的基部类群，具有与唇形目两侧对称花早期分化相关的各种两侧对称花类型。因此，在苦苣苔科选择两侧对称花代表类群开展花对称性的进化发育生物学研究十分有助于探讨苦苣苔科花对称性基因在两侧对称花类群的调控进化，从而揭示广义唇形目基部类群花对称性的进化发育模式。我们选择苦苣苔科两侧对称花类群烟叶唇柱苣苔（Chirita heterotricha）为主要研究材料。烟叶唇柱苣苔是苦苣苔科两侧对称性比较强烈的类群，主要体现在其只有腹部两枚雄蕊能育，背部和侧部雄蕊均败育，与金鱼草的近亲沙漠幽灵花（Mohaveae confertiflora）的形态类似。在沙漠幽灵花中CYC 类基因的表达从背部延伸到了侧部，但是在烟叶唇柱苣苔中是否遵循同一模式还是存在其他途径这是我们的主要研究目的。此外，我们发现在烟叶唇柱苣苔花序的顶花中偶尔会发生腹部化的辐射对称突变花。在金鱼草和柳穿鱼中，CYC 类基因的失活导致了辐射对称突变花的产生，而在豆科植物Cadia purpurea 中，CYC 类基因的活性从背部延伸到了侧部和腹部，导致了辐射对称花的形成。烟叶唇柱苣苔中的情形则值得我们关注。再者，革叶粗筒苣苔（Briggisia mihieri）是烟叶唇柱苣苔的近缘类群，但它具有四枚能育雄蕊（侧部和腹部各两枚）。苦苣苔科两能育雄蕊类群被认为起源于四能育雄蕊类群。我们在烟叶唇柱苣苔野生型两侧对称花和辐射对称突变花中开展花对称性基因的表达模式研究，结合在革叶粗筒苣苔中的研究，试图揭示烟叶唇柱苣苔两雄蕊类两侧对称花形成的分子机制和进化发育途径。 本研究从烟叶唇柱苣苔中分离到4 个CYC 类基因（ChCYC1C，ChCYC1D，ChCYC2A，ChCYC2B），2 个DIV 类基因（ChDIV1，ChDIV2）和2 个RAD 类基因（ChRAD1，ChRAD2）。从革叶粗筒苣苔中分离到4 个CYC 类基因（BmCYC1C，BmCYC1D，BmCYC2A，BmCYC2B），2 个DIV 类基因（BmDIV1，BmDIV2）和1 个RAD 类基因（BmRAD1）。序列和系统发育分析结果显示它们均为花对称性基因。进一步的表达分析显示结果表明：（1）不同拷贝CYC 类基因的表达存在着分化，与其氨基酸序列的分化一致；烟叶唇柱苣苔的CYC 类基因表达从背部延伸到了侧部而革叶粗筒苣苔的CYC 类基因仅在背部表达，与它们的形态分化密切相关。（2）DIV 类基因在种内两个拷贝以及种间的表达无明显的分化，在花的各个部位都有表达。（3）烟叶唇柱苣苔两个拷贝的RAD 类基因的表达存在分化；两个种的同类RAD 类基因的表达也存在分化。这些花对称性基因的表达模式分析揭示了它们在烟叶唇柱苣苔和革叶粗筒苣苔两侧对称花形态建成以及二者花形态的演化中具有重要作用。 同时，我们对烟叶唇柱苣苔的辐射对称突变花进行了自交和F1 代培育，但是F1 代没有出现稳定的辐射对称突变株。我们从辐射对称突变花中分离到的CYC 类、DIV 类和RAD 类基因的核苷酸序列与野生型的完全一致。说明突变体的产生与序列变异无关。RT-PCR 分析表明突变花中CYC 类基因全部失活，而DIV 类基因和在腹部表达的RAD 类基因的表达信号有所增强，这与其腹部化辐射对称的形态相一致。这进一步证实了花对称性基因在烟叶唇柱苣苔花形态建成中的作用。

The role of MADS and TCP transcription factors in Gerbera hybrida flower development

Angiosperms represent a huge diversity in floral structures. Thus, they provide an attractive target for comparative developmental genetics studies. Research on flower development has focused on few main model plants, and studies on these species have revealed the importance of transcription factors, such as MADS-box and TCP genes, for regulating the floral form. The MADS-box genes determine floral organ identities, whereas the TCP genes are known to regulate flower shape and the number of floral organs. In this study, I have concentrated on these two gene families and their role in regulating flower development in Gerbera hybrida, a species belonging to the large sunflower family (Asteraceae). The Gerbera inflorescence is comprised of hundreds of tightly clustered flowers that differ in their size, shape and function according to their position in the inflorescence. The presence of distinct flower types tells Gerbera apart from the common model species that bear only single kinds of flowers in their inflorescences. The marginally located ray flowers have large bilaterally symmetrical petals and non-functional stamens. The centrally located disc flowers are smaller, have less pronounced bilateral symmetry and carry functional stamens. Early stages of flower development were studied in Gerbera to understand the differentiation of flower types better. After morphological analysis, we compared gene expression between ray and disc flowers to reveal transcriptional differences in flower types. Interestingly, MADS-box genes showed differential expression, suggesting that they might take part in defining flower types by forming flower-type-specific regulatory complexes. Functional analysis of a CYCLOIDEA-like TCP gene GhCYC2 provided evidence that TCP transcription factors are involved in flower type differentiation in Gerbera. The expression of GhCYC2 is ray-flower-specific at early stages of development and activated only later in disc flowers. Overexpression of GhCYC2 in transgenic Gerbera-lines causes disc flowers to obtain ray-flower-like characters, such as elongated petals and disrupted stamen development. The expression pattern and transgenic phenotypes further suggest that GhCYC2 may shape ray flowers by promoting organ fusion. Cooperation of GhCYC2 with other Gerbera CYC-like TCP genes is most likely needed for proper flower type specification, and by this means for shaping the elaborate inflorescence structure. Gerbera flower development was also approached by characterizing B class MADS-box genes, which in the main model plants are known regulators of petal and stamen identity. The four Gerbera B class genes were phylogenetically grouped into three clades; GGLO1 into the PI/GLO clade, GDEF2 and GDEF3 into the euAP3 clade and GDEF1 into the TM6 clade. Putative orthologs for GDEF2 and GDEF3 were identified in other Asteraceae species, which suggests that they appeared through an Asteraceae-specific duplication. Functional analyses indicated that GGLO1 and GDEF2 perform conventional B-function as they determine petal and stamen identities. Our studies on GDEF1 represent the first functional analysis of a TM6-like gene outside the Solanaceae lineage and provide further evidence for the role of TM6 clade members in specifying stamen development. Overall, the Gerbera B class genes showed both commonalities and diversifications with the conventional B-function described in the main model plants.

1921 annotations to Corpus juris : 1-22 C.J. ; and Workmen's compensation acts third volume permanent cyc annotations : contains annotations from all cases decided since the original treatises were written ; also errata corrected, new principles announced, etc. /

On cover: Annotations, Corpus juris-Cyc service. Permanent volume. 1921. C.J., A-Ev ; Cyc, Ex-Z.

Contrast sensitivity changes due to glaucoma and normal aging: low-spatial-frequency losses in both magnocellular and parvocellular pathways

PURPOSE: To explore the effects of glaucoma and aging on low-spatial-frequency contrast sensitivity by using tests designed to assess performance of either the magnocellular (M) or parvocellular (P) visual pathways. METHODS: Contrast sensitivity was measured for spatial frequencies of 0.25 to 2 cyc/deg by using a published steady- and pulsed-pedestal approach. Sixteen patients with glaucoma and 16 approximately age-matched control subjects participated. Patients with glaucoma were tested foveally and at two midperipheral locations: (1) an area of early visual field loss, and (2) an area of normal visual field. Control subjects were assessed in matched locations. An additional group of 12 younger control subjects (aged 20-35 years) were also tested. RESULTS: Older control subjects demonstrated reduced sensitivity relative to the younger group for the steady (presumed M)- and pulsed (presumed P)-pedestal conditions. Sensitivity was reduced foveally and in the midperiphery across the spatial frequency range. In the area of early visual field loss, the glaucoma group demonstrated further sensitivity reduction relative to older control subjects across the spatial frequency range for both the steady- and pulsed-pedestal tasks. Sensitivity was also reduced in the midperipheral location of "normal" visual field for the pulsed condition. CONCLUSIONS: Normal aging results in a reduction of contrast sensitivity for the low-spatial-frequency-sensitive components of both the M and P pathways. Glaucoma results in a further reduction of sensitivity that is not selective for M or P function. The low-spatial-frequency-sensitive channels of both pathways, which are presumably mediated by cells with larger receptive fields, are approximately equivalently impaired in early glaucoma.

Dopamine and visually regulated eye growth in chick

Retinal image properties such as contrast and spatial frequency play important roles in the development of normal vision. For example, visual environments comprised solely of low contrast and/or low spatial frequencies induce myopia. The visual image is processed by the retina and it then locally controls eye growth. In terms of the retinal neurotransmitters that link visual stimuli to eye growth, there is strong evidence to suggest involvement of the retinal dopamine (DA) system. For example, effectively increasing retinal DA levels by using DA agonists can suppress the development of form-deprivation myopia (FDM). However, whether visual feedback controls eye growth by modulating retinal DA release, and/or some other factors, is still being elucidated. This thesis is chiefly concerned with the relationship between the dopaminergic system and retinal image properties in eye growth control. More specifically, whether the amount of retinal DA release reduces as the complexity of the image degrades was determined. For example, we investigated whether the level of retinal DA release decreased as image contrast decreased. In addition, the effects of spatial frequency, spatial energy distribution slope, and spatial phase on retinal DA release and eye growth were examined. When chicks were 8-days-old, a cone-lens imaging system was applied monocularly (+30 D, 3.3 cm cone). A short-term treatment period (6 hr) and a longer-term treatment period (4.5 days) were used. The short-term treatment tests for the acute reduction in DA release by the visual stimulus, as is seen with diffusers and lenses, whereas the 4.5 day point tests for reduction in DA release after more prolonged exposure to the visual stimulus. In the contrast study, 1.35 cyc/deg square wave grating targets of 95%, 67%, 45%, 12% or 4.2% contrast were used. Blank (0% contrast) targets were included for comparison. In the spatial frequency study, both sine and square wave grating targets with either 0.017 cyc/deg and 0.13 cyc/deg fundamental spatial frequencies and 95% contrast were used. In the spectral slope study, 30% root-mean-squared (RMS) contrast fractal noise targets with spectral fall-off of 1/f0.5, 1/f and 1/f2 were used. In the spatial alignment study, a structured Maltese cross (MX) target, a structured circular patterned (C) target and the scrambled versions of these two targets (SMX and SC) were used. Each treatment group comprised 6 chicks for ocular biometry (refraction and ocular dimension measurement) and 4 for analysis of retinal DA release. Vitreal dihydroxyphenylacetic acid (DOPAC) was analysed through ion-paired reversed phase high performance liquid chromatography with electrochemical detection (HPLC-ED), as a measure of retinal DA release. For the comparison between retinal DA release and eye growth, large reductions in retinal DA release possibly due to the decreased light level inside the cone-lens imaging system were observed across all treated eyes while only those exposed to low contrast, low spatial frequency sine wave grating, 1/f2, C and SC targets had myopic shifts in refraction. Amongst these treatment groups, no acute effect was observed and longer-term effects were only found in the low contrast and 1/f2 groups. These findings suggest that retinal DA release does not causally link visual stimuli properties to eye growth, and these target induced changes in refractive development are not dependent on the level of retinal DA release. Retinal dopaminergic cells might be affected indirectly via other retinal cells that immediately respond to changes in the image contrast of the retinal image.

Early time course of Akt phosphorylation after endurance and resistance exercise

Purpose The aim of this study was to determine the early time course of exercise-induced signaling after divergent contractile activity associated with resistance and endurance exercise. Methods Sixteen male subjects were randomly assigned to either a cycling (CYC; n = 8, 60 min, 70% V?O2peak) or resistance (REX; n = 8, 8×5 leg extension, 80% one-repetition maximum, 3-min recovery) exercise group. Serial muscle biopsies were obtained from vastus lateralis at rest before, immediately after, and after 15, 30, and 60 min of passive recovery to determine early signaling responses after exercise. Results There were comparable increases from rest in AktThr308/Ser473 and mTORSer2448 phosphorylation during the postexercise time course that peaked 30-60 min after both CYC and REX (P<0.05). There were also similar patterns in p70S6K Thr389 and 4E-BP1Thr37/46 phosphorylation, but a greater magnitude of effect was observed for REX and CYC, respectively (P<0.05). However, AMPKThr172 phosphorylation was only significantly elevated after CYC (P<0.05), and we observed divergent responses for glycogen synthaseSer641 and AS160 phosphorylation that were enhanced after CYC but not REX (P<0.05). Conclusions We show a similar time course for Akt-mTOR-S6K phosphorylation during the initial 60-min recovery period after divergent contractile stimuli. Conversely, enhanced phosphorylation status of proteins that promote glucose transport and glycogen synthesis only occurred after endurance exercise. Our results indicate that endurance and resistance exercise initiate translational signaling, but high-load, low-repetition contractile activity failed to promote phosphorylation of pathways regulating glucose metabolism.

Chiral Synthons from Monoterpenes.1 Stereoselective Syntheses of (-)5S, 6S, 9R-6-Isopropyl-9-Methyl-2-Oxaspiro[4.4]Nonan-3-One and (-) 3aR, 6R, 7aR-3a-Methyl-6-Isopropenylhexahydrobenzofuran-2-One

Highly stereoselective syntheses of two C-12 chiral synthons 3 and 9, mentioned in the title, starting from the monoterpenes R-1 imonene and R-carvone, using radical cyc 1 sation as key reaction, are described.

Studies on LDPE-cyanoethylated lignocellulosics blends using epoxy functionalized LDPE as compatibilizer

Rubberwood flour and cellulose have been plasticized by cyanoethylation and then blended with low-density polyethylene (LDPE). A small quantity of epoxy functionalized polyethylene i.e., polyethylene-co-glycidyl methacrylate (PEGMA) has been added to further enhance the mechanical properties. The mechanical properties were measured according to the standard ASTM methods. SEM analysis was performed for both fractured and unfractured blend specimens. The mechanical properties were improved by the addition of PEGMA compatibilizer. LDPE blends with cyanoethylated wood flour (CYWF) showed higher tensile strength and modulus than cyanoethylated cellulose CYC-LDPE blends. However CYC-LDPE blends exhibited higher relative elongation at break values as compared with the former. The TGA analysis showed lowering of thermal stability as the filler content is increased and degradation temperature of LDPE is shifted slightly to lower temperature. DSC analysis showed loss of crystallinity for the LDPE phase as the filler content is increased for both types of blends. Dielectric properties of the blends were similar to LDPE, but were lowered on adding PEGMA. (c) 2006 Wiley Periodicals, Inc.