DOPAMINE D-1 RECEPTOR MECHANISMS IN THE COGNITIVE PERFORMANCE OF YOUNG-ADULT AND AGED MONKEYS

Dopamine (DA) D-1 receptor compounds were examined in monkeys for effects on the working memory functions of the prefrontal cortex and on the fine motor abilities of the primary motor cortex. The D-1 antagonist, SCH23390, the partial D-1 agonist, SKF38393, and the full D-1 agonist, dihydrexidine, were characterized in young control monkeys, and in aged monkeys with naturally occurring catecholamine depletion. In addition, SKF38393 was tested in young monkeys experimentally depleted of catecholamines with chronic reserpine treatment. Injections of SCH23390 significantly impaired the memory performance of young control monkeys, but did not impair aged monkeys with presumed catecholamine depletion. Conversely, the partial agonist, SKF38393, improved the depleted monkeys (aged or reserpine-treated) but did not improve young control animals. The full agonist, dihydrexidine, did improve memory performance in young control monkeys, as well as in a subset of aged monkeys. Consistent with D, receptor mechanisms, agonist-induced improvements were blocked by SCH23390. Drug effects on memory performance occurred independently of effects on fine motor performance. These results underscore the importance of DA D-1 mechanisms in cognitive function, and provide functional evidence of DA system degeneration in aged monkeys. Finally, high doses of D-1 agonists impaired memory performance in aged monkeys, suggesting that excessive D-1 stimulation may be deleterious to cognitive function.

Dose-dependent effects of the dopamine D1 receptor agonists A77636 or SKF81297 on spatial working memory in aged monkeys

With advancing age, monkeys develop deficits in spatial working memory resembling those induced by lesions of the prefrontal cortex (PFC). Aged monkeys also exhibit marked loss of dopamine from the PFC, a transmitter known to be important for proper PFC cognitive function. Previous results suggest that D1 agonist treatment can improve spatial working memory abilities in aged monkeys. However, this research was limited by the use of drugs with either partial agonist actions or significant D2 receptor actions. In our study, the selective dopamine D1 receptor full agonists A77636 and SKF81297 were examined in aged monkeys for effects on the working memory functions of the PFC. Both compounds produced a significant, dose-related effect on delayed response performance without evidence of side effects: low doses improved performance although higher doses impaired or had no effect on performance. Both the improvement and impairment in performance were reversed by pretreatment with the D1 receptor antagonist, SCH23390. These findings are consistent with previous results demonstrating that there is a narrow range of D1 receptor stimulation for optimal PFC cognitive function, and suggest that very low doses of D1 receptor agonists may have cognitive-enhancing actions in the elderly.

Improving effects of huperzine A on spatial working memory in aged monkeys and young adult monkeys with experimental cognitive impairment

Our previous studies demonstrated that huperzine A, a reversible and selective acetylcholinesterase inhibitor, exerts beneficial effects on memory deficits in various rodent models of amnesia. To extend the antiamnesic action of huperzine A to nonhuman primates, huperzine A was evaluated for its ability to reverse the deficits in spatial memory produced by scopolamine in young adult monkeys or those that are naturally occurring in aged monkeys using a delayed-response task. Scopolamine, a muscarinic receptor antagonist, dose dependently impaired performance with the highest dose (0.03 mg/kg, i.m.) producing a significant reduction in choice accuracy in young adult monkeys. The delayed performance changed from an average of 26.8/30 trials correct on saline control to an average of 20.2/30 trials correct after scopolamine administration. Huperzine A (0.01-0.1 mg/kg, i.m.) significantly reversed deficits induced by scopolamine in young adult monkeys on a delayed-response task; performance after an optimal dose (0.1 mg/kg) averaged 25.0/30 correct. In four aged monkeys, huperzine A (0.001-0.01 mg/kg, i.m.) significantly increased choice accuracy from 20.5/30 on saline control to 25.2/30 at the optimal dose (0.001 mg/kg for two monkeys and 0.01 mg/kg for the other two monkeys). The beneficial effects of huperzine A on delayed-response performance were long lasting; monkeys remained improved for about 24 h after a single injection of huperzine A. This study extended the findings that huperzine A improves the mnemonic performance requiring working memory in monkeys, and suggests that huperzine A may be a promising agent for clinical therapy of cognitive impairments in patients with Alzheimer's disease.

鱼类耳石微结构的计算机自动识别

本文介绍了鱼类耳石微结构图像识别系统的工作原理和应用实例 ,该系统实现了对鱼类的耳石日轮的自动识别计数和测量日轮宽度 ,并将分析结果自动保存为Excel可识别格式的数据文件。用草鱼微耳石对该系统进行测试的结果表明 ,在随机抽取的 30个样本中 ,有 2 8个样本日轮自动识别的正确率为 1 0 0 % ,其余 2个样本经过手工修改后也能达到对日轮的完全识别。此外 ,采用 768× 5 82像素的BMP格式数字图像测量日轮宽度 ,计算机的分辨率比显微镜提高了约 1 3倍。该系统还可应用于鱼类的鳞片、胸鳍棘和脊

Measuring EGFR Separations on Cells with ∼10 nm Resolution via Fluorophore Localization Imaging with Photobleaching

Detecting receptor dimerisation and other forms of clustering on the cell surface depends on methods capable of determining protein-protein separations with high resolution in the ∼10-50 nm range. However, this distance range poses a significant challenge because it is too large for fluorescence resonance energy transfer and contains distances too small for all other techniques capable of high-resolution in cells. Here we have adapted the technique of fluorophore localisation imaging with photobleaching to measure inter-receptor separations in the cellular environment. Using the epidermal growth factor receptor, a key cancer target molecule, we demonstrate ∼10 nm resolution while continuously covering the range of ∼10-80 nm. By labelling the receptor on cells expressing low receptor numbers with a fluorescent antagonist we have found inter-receptor separations all the way up from 8 nm to 59 nm. Our data are consistent with epidermal growth factor receptors being able to form homo-polymers of at least 10 receptors in the absence of activating ligands. © 2013 Needham et al.

Hexabromocyclododecane-induced developmental toxicity and apoptosis in zebrafish embryos

Hexabromocyclododecane (HBCD) is widely used as a brominated flame retardant, and has been detected in the aquatic environment, wild animals, and humans. However, details of the environmental health risk of HBCD are not well known. In this study, zebrafish embryos were used to assess the developmental toxicity of the chemical. Four-hour post-fertilization (hpf) zebrafish embryos were exposed to various concentrations of HBCD (0, 0.05, 0.1, 0.5, and 1.0 mg L-1) until 96 h. Exposure to 0.1, 0.5, and 1.0 mg L-1 HBCD significantly increased the malformation rate and reduced survival in the 0.5 and 1.0 mg L-1 HBCD exposure groups. Acridine orange (AO) staining showed that HBCD exposure resulted in cell apoptosis. Reactive oxygen species (ROS) was significantly induced at exposures of 0.1, 0.5, and 1.0 mg L-1 HBCD. To test the apoptotic pathway, several genes related to cell apoptosis, such as p53, Puma, Apaf-1, caspase-9, and caspase-3, were examined using real-time PCR. The expression patterns of these genes were up-regulated to some extent. Two anti-apoptotic genes, Mdm2 (antagonist of p53) and Bcl-2 (inhibitor of Bax), were down-regulated, and the activity of capspase-9 and caspase-3 was significantly increased. The overall results demonstrate that waterborne HBCD is able to produce oxidative stress and induce apoptosis through the involvement of caspases in zebrafish embryos. The results also indicate that zebrafish embryos can serve as a reliable model for the developmental toxicity of HBCD. (C) 2009 Elsevier B.V. All rights reserved.

Effects of adrenergic agents on the expression of zebrafish (Danio rerio) vitellogenin Ao1

Teleost vitellogenins (VTGs) are large multidomain apolipoproteins, traditionally considered to be estrogen-responsive precursors of the major egg yolk proteins, expressed and synthesized mainly in hepatic tissue. The inducibility of VTGs has made them one of the most frequently used in vivo and in vitro biomarkers of exposure to estrogen-active substances. A significant level of zebrafish vtgAo1, a major estrogen responsive form, has been unexpectedly found in heart tissue in our present studies. Our studies on zebrafish cardiomyopathy, caused by adrenergic agonist treatment, suggest a similar protective function of the cardiac expressed vtgAo1. We hypothesize that its function is to unload surplus intracellular lipids in cardiomyocytes for "reverse triglyceride transportation" similar to that found in lipid transport proteins in mammals. Our results also demonstrated that zebrafish vtgAo1 mRNA expression in heart can be suppressed by both (x-adrenergic agonist, phenylephrine (PE) and beta-adrenergic agonist, isoproterenol (ISO). Furthermore, the strong stimulation of zebrafish vtgAo1 expression in plasma induced by the beta-adrenergic antagonist, MOXIsylyl, was detected by Enzyme-Linked ImmunoSorbent Assay (ELISA). Such stimulation cannot be suppressed by taMOXIfen, an antagonist to estrogen receptors. Thus, Our present data indicate that the production of teleost VTG in vivo can be regulated not only by estrogenic agents, but by adrenergic signals as well. (c) 2009 Elsevier Inc. All rights reserved.

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.

DE-71-induced apoptosis involving intracellular calcium and the Bax-mitochondria-caspase protease pathway in human neuroblastoma cells In vitro

Polybrominated diphenyl ethers (PBDEs) are used extensively as flame-retardants and are ubiquitous in the environment and in wildlife and human tissue. Recent studies have shown that PBDEs induce neurotoxic effects in vivo and apoptosis in vitro. However, the signaling mechanisms responsible for these events are still unclear. In this study, we investigated the action of a commercial mixture of PBDEs (pentabrominated diphenyl ether, DE-71) on a human neuroblastoma cell line, SK-N-SH. A cell viability test showed a dose-dependent increase in lactate dehydrogenase leakage and 3-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyl-tetrazolium bromide reduction. Cell apoptosis was observed through morphological examination, and DNA degradation in the cell cycle and cell apoptosis were demonstrated using flow cytometry and DNA laddering. The formation of reactive oxygen species was not observed, but DE-71 was found to significantly induce caspase-3, -8, and -9 activity, which suggests that apoptosis is not induced by oxidative stress but via a caspase-dependent pathway. We further investigated the intracellular calcium ([Ca2+](i)) levels using flow cytometry and observed an increase in the intracellular Ca2+ concentration with a time-dependent trend. We also found that the N-methyl d-aspartate (NMDA) receptor antagonist MK801 (3 mu M) significantly reduced DE-71-induced cell apoptosis. The results of a Western blotting test demonstrated that DE-71 treatment increases the level of Bax translocation to the mitochondria in a dose-dependent fashion and stimulates the release of cytochrome c (Cyt c) from the mitochondria into the cytoplasm. Overall, our results indicate that DE-71 induces the apoptosis of ([Ca2+](i)) in SK-N-SH cells via Bax insertion, Cyt c release in the mitochondria, and the caspase activation pathway.

Self-organization of reflexive behavior from spontaneous motor activity.

In mammals, the development of reflexes is often regarded as an innate process. However, recent findings show that fetuses are endowed with favorable conditions for ontogenetic development. In this article, we hypothesize that the circuitry of at least some mammalian reflexes can be self-organized from the sensory and motor interactions brought forth in a musculoskeletal system. We focus mainly on three reflexes: the myotatic reflex, the reciprocal inhibition reflex, and the reverse myotatic reflex. To test our hypothesis, we conducted a set of experiments on a simulated musculoskeletal system using pairs of agonist and antagonist muscles. The reflex connectivity is obtained by producing spontaneous motor activity in each muscle and by correlating the resulting sensor and motor signals. Our results show that, under biologically plausible conditions, the reflex circuitry thus obtained is consistent with that identified in relation to the analogous mammalian reflexes. In addition, they show that the reflex connectivity obtained depends on the morphology of the musculoskeletal system as well as on the environment that it is embedded in.

Evaluation of soot particles of biomass fuels with endocrine-modulating activity in yeast-based bioassay

Exposure to indoor air pollution (IAP) from the combustion of biomass fuels is an important cause of morbidity and mortality in developing countries. In the work discussed in this paper we evaluated the endocrine activity of soot particles from biomass fuels by using yeast bioassay. These pollutants could have beta-galactosidase activity with a relative potency (RP) about 10(-7)-10(-9) that of estradiol. Soot particles from wood and straw combustion only partially induced beta-galactosidase activity whereas others produced fully inductive activity in the yeast assay system. These pollutants did not have estrogen antagonist and progesterone agonist activity within the defined concentration range. However, these pollutants require 2-4 orders of magnitude higher IC50 to inhibit the activity of progesterone in a similar dose-response manner to mifepristone. We therefore propose that the endocrine activity of some environmental pollutants may be because of inhibition of the progesterone receptor (hPR). GC-MS results showed that substituted polycyclic aromatic hydrocarbon (PAH) compounds, substituted phenolic compounds and derivatives, aromatic carbonyl compounds, and phytosteroids in these soot particles may be mimicking endogenous hormones.

符合国际与国家标准的藏文Linux操作系统研发

藏文操作系统是实施藏文信息处理、实现藏文信息化的基础。介绍了藏文信息处理的发展现状,并分析了藏文信息处理的需求。阐述了研发藏文操作系统涉及到的主要研究内容,以Linux操作系统模块结构为基础阐述了该系统采用的实现框架,分析了其中需要解决的关键技术难点,详细描述其解决方案。简要说明了"藏文Linux桌面系统V1.1"的特点及后续的工作方向。

非洲爪蟾Nkx6.3在原肠运动和神经嵴诱导调控中的功能及爪蟾神经管腹侧图式形成的研究

神经嵴（neural crest）是一类脊椎动物特有的多潜能迁移细胞。这一类细胞历经“表皮—间充质”转换（EMT），与神经管背侧的其它细胞分离，经由不同路线迁移，定位于胚胎外周各处，后分化为不同的细胞类型包括外周神经系统、颅面骨骼系统及色素细胞等。神经嵴的发育是一个多途径多步骤的过程，受多种信号通路及转录因子调控。这些调控因子相互调节形成精密网络，可被划分为三个主要层次类群：分泌性信号分子（BMP、Wnt、FGF、Delta）、神经板边界特异基因（Msx、Pax3/7、 Zic1、Dlx3/5）、神经嵴特异基因（Snail/Slug、AP-2、FoxD3、Twist、Id、cMyc、Sox9/10）。本文第一章主要概述不同组织来源的各种分泌信号在神经嵴诱导中的作用以及他们之间的整合调控。 Nkx6家族蛋白是一类进化上保守的转录因子，在脊椎动物中枢神经系统（CNS)的图式形成和胰腺的发育中有重要作用。在第二章，我们描述了非洲爪蟾中Nkx6家族基因的克隆及其表达图式。与小鼠和鸡中的同源基因类似，爪蟾的Nkx6家族基因在胚胎发育过程中主要表达于中枢神经系统和前部内胚层组织。其中Nkx6.1和Nkx6.2在神经胚期神经板表达重合，晚期都表达于后脑和脊髓的腹侧。Nkx6.3从卵裂期到神经胚早期都表达于非神经外胚层，而尾芽期表达于后脑后部和腮弓。在内胚层中，Nkx6.2在尾芽期表达于底索。在蝌蚪期，Nkx6家族的三个基因分别表达于前部内胚层的衍生物，包括胰腺、胃、食道和肺。 Nkx6.3是最近发现的Nkx6家族新成员，它在爪蟾中的表达与Nkx6.1和Nkx6.2有了较大分歧。在第三章，我们通过功能获得及功能缺失实验来探讨Nkx6.3在爪蟾早期发育中的功能。我们发现原肠期前过量或抑制Nkx6.3表达都会影响胚胎原肠运动的正常进行。我们通过动物帽延伸实验证明Nkx6.3参与了细胞运动。半定量RT-PCR结果显示，Nkx6.3可以调控一些粘附分子的表达。以上结果说明Nkx6.3通过调控粘附分子的转录而参与细胞运动的调控。我们还发神经嵴（neural crest）是一类脊椎动物特有的多潜能迁移细胞。这一类细胞历经“表皮—间充质”转换（EMT），与神经管背侧的其它细胞分离，经由不同路线迁移，定位于胚胎外周各处，后分化为不同的细胞类型包括外周神经系统、颅面骨骼系统及色素细胞等。神经嵴的发育是一个多途径多步骤的过程，受多种信号通路及转录因子调控。这些调控因子相互调节形成精密网络，可被划分为三个主要层次类群：分泌性信号分子（BMP、Wnt、FGF、Delta）、神经板边界特异基因（Msx、Pax3/7、 Zic1、Dlx3/5）、神经嵴特异基因（Snail/Slug、AP-2、FoxD3、Twist、Id、cMyc、Sox9/10）。本文第一章主要概述不同组织来源的各种分泌信号在神经嵴诱导中的作用以及他们之间的整合调控。 Nkx6家族蛋白是一类进化上保守的转录因子，在脊椎动物中枢神经系统（CNS)的图式形成和胰腺的发育中有重要作用。在第二章，我们描述了非洲爪蟾中Nkx6家族基因的克隆及其表达图式。与小鼠和鸡中的同源基因类似，爪蟾的Nkx6家族基因在胚胎发育过程中主要表达于中枢神经系统和前部内胚层组织。其中Nkx6.1和Nkx6.2在神经胚期神经板表达重合，晚期都表达于后脑和脊髓的腹侧。Nkx6.3从卵裂期到神经胚早期都表达于非神经外胚层，而尾芽期表达于后脑后部和腮弓。在内胚层中，Nkx6.2在尾芽期表达于底索。在蝌蚪期，Nkx6家族的三个基因分别表达于前部内胚层的衍生物，包括胰腺、胃、食道和肺。 Nkx6.3是最近发现的Nkx6家族新成员，它在爪蟾中的表达与Nkx6.1和Nkx6.2有了较大分歧。在第三章，我们通过功能获得及功能缺失实验来探讨Nkx6.3在爪蟾早期发育中的功能。我们发现原肠期前过量或抑制Nkx6.3表达都会影响胚胎原肠运动的正常进行。我们通过动物帽延伸实验证明Nkx6.3参与了细胞运动。半定量RT-PCR结果显示，Nkx6.3可以调控一些粘附分子的表达。以上结果说明Nkx6.3通过调控粘附分子的转录而参与细胞运动的调控。我们还发现，在爪蟾胚胎中Nkx6.3的过表达或抑制表达都导致神经嵴标记基因表达降低。进一步研究发现，32细胞期在不同部位注射Nkx6.3 mRNA可以异位诱导或抑制Slug的表达。动物帽实验显示，Nkx6.3单独过表达可以诱导神经嵴发生，而迄今为止转录因子中只有Snail1具有这一单独诱导能力。在爪蟾胚胎及动物帽中，过表达Nkx6.3都可以诱导Fgf8、Wnt8而抑制BMP4的转录，而且Nkx6.3对这些分泌因子的调控方式是不同的。4细胞期过表达Nkx6.3的胚胎，在促进Fgf8和Wnt8而抑制BMP4的同时，却抑制神经板边界特异基因Msx1、Pax3和神经嵴特异基因Slug的表达，说明Nkx6.3对神经嵴的诱导调控在神经板边界基因层次还存在抑制作用。32细胞过表达Nkx6.3会细胞自主性抑制以及细胞非自主诱导Msx1、Pax3、Slug的表达。Nkx6.3异位诱导Dlx5却抑制Dlx3的表达，说明Dlx5可能是Nkx6.3负调控的直接靶基因。由此，我们提出Nkx6.3的神经嵴诱导调控分为两个层次：分泌信号分子水平的正调控和神经板边界决定水平的负调控。在脊椎动物的神经发生过程中，神经管背腹不同层次形成不同的神经元。这些神经元细胞的命运由背腹起源的多种形态发生素决定。形态发生素通过浓度梯度确定了一组转录因子在神经管背腹不同层次的特异表达，这些基因的组合调控决定了神经前体细胞的命运。然而，这些转录因子是如何解读形态发生素梯度信号的还不是很清楚。第四章，我们通过对神经管腹侧特异表达的转录因子的调控区进行预测，确定了可能调控这些基因表达的保守区段。此外，我们改进了爪蟾转基因操作，并用这一技术确证了Nkx6.2的调控区域。Dbx1、Nkx2.2及Pax6的转录调控区已在小鼠或爪蟾中报道过。由此我们得到了两对在神经管背腹图式中相互作用的转录因子的调控区域：Nkx6.2和Dbx1、Nkx2.2和Pax6。通过对Nkx6.2和Dbx1的调控保守区的转录因子结合位点的预测，我们发现这四个基因以及Wnt信号之间存在大量的相互调控。然而在这两个基因的调控区，我们没有发现Gli的调控位点，暗示这两个基因可能不受Shh的直接调控。我们还克隆了Dbx家族的两个基因，并检测了它们的时空特异性表达，发现Dbx2是母源性表达的，而Dbx1是合子型基因。这两个基因的表达图式相似，都在神经板中线两侧成线状表达，尾芽期在神经管中部表达。过表达Dbx2抑制神经元的初级分化，说明它可能与Dbx1一样具有维持神经板细胞未分化状态的功能。Dbx2的过表达还抑制Nkx6.2及Dbx1的表达，说明它们可能一起参与了神经管腹侧图式的调控。

非洲爪蛙分泌型叶酸结合蛋白及NKX6.3在早期胚胎神经发育中的功能研究

叶酸是B族维生素的一员，参与体内一系列重要的生命过程包括DNA，氨基酸的合成，调控细胞周期，参与一碳单位供体循环，调节DNA,蛋白质甲基化等。叶酸的许多功能都和叶酸结合蛋白有关，体内有多种跨膜形式的叶酸结合蛋白，比如Folbp1，RFC，HCP等。以前的研究表明这些不同的叶酸结合蛋白具有不同的功能。分泌型叶酸结合蛋白是另外一类叶酸结合蛋白，在人类，小鼠，猪中都有序列报道，但是其功能却知之甚少。 我们在非洲爪蛙中鉴定出一个全新的分泌型叶酸结合蛋白并命名为Secreted Folate Binding Protein(sFBP)。在胚胎和转染细胞系中我们都证明该蛋白是分泌性的，表面等离子共振实验发现sFBP能够结合叶酸。在胚胎早期这个基因表达于粘液腺和神经板区域，神经管闭合后在神经管、粘液腺、眼睛，头部以及鳃弓都有表达。特异morpholino 阻断sFBP翻译后发现粘液腺发育异常，神经管闭合缺陷，前后体轴聚集延伸运动受到抑制，尾芽期胚胎表现出体轴缩短，无眼，小头或无头的表型。进一步研究发现显微注射sFBP morpholino 的胚胎神经板区域细胞发生凋亡，中胚层和神经外胚层的一系列粘附分子表达异常，神经细胞的正常分化也受到抑制。通过显微移植实验我们还发现抑制sFBP的翻译后，神经嵴细胞的正常分化和迁移都受到抑制。但是，显微注射叶酸及其类似物或者显微注射甲基供体S-腺苷甲硫氨酸或者亮氨酸甲基转移酶都不能挽救阻断sFBP造成的表形，由此提示sFBP可能不是通过叶酸传统的参与营养合成或者甲基化的途径发挥作用。我们发现注射sFBP morpholino可以抑制Islet-1mRNA和蛋白质的表达，Islet-1的表达区域与sFBP类似。共同注射Islet-1 mRNA和sFBP morpholino可以极大的挽救sFBP morpholino的表型。最后通过morpholino特异阻断Islet-1的表达后，我们发现其表现出与sFBP morpholino类似的粘液腺发育缺陷，神经板细胞凋亡，小头无眼的表形。由此叶酸是B族维生素的一员，参与体内一系列重要的生命过程包括DNA，氨基酸的合成，调控细胞周期，参与一碳单位供体循环，调节DNA,蛋白质甲基化等。叶酸的许多功能都和叶酸结合蛋白有关，体内有多种跨膜形式的叶酸结合蛋白，比如Folbp1，RFC，HCP等。以前的研究表明这些不同的叶酸结合蛋白具有不同的功能。分泌型叶酸结合蛋白是另外一类叶酸结合蛋白，在人类，小鼠，猪中都有序列报道，但是其功能却知之甚少。 我们在非洲爪蛙中鉴定出一个全新的分泌型叶酸结合蛋白并命名为Secreted Folate Binding Protein(sFBP)。在胚胎和转染细胞系中我们都证明该蛋白是分泌性的，表面等离子共振实验发现sFBP能够结合叶酸。在胚胎早期这个基因表达于粘液腺和神经板区域，神经管闭合后在神经管、粘液腺、眼睛，头部以及鳃弓都有表达。特异morpholino 阻断sFBP翻译后发现粘液腺发育异常，神经管闭合缺陷，前后体轴聚集延伸运动受到抑制，尾芽期胚胎表现出体轴缩短，无眼，小头或无头的表型。进一步研究发现显微注射sFBP morpholino 的胚胎神经板区域细胞发生凋亡，中胚层和神经外胚层的一系列粘附分子表达异常，神经细胞的正常分化也受到抑制。通过显微移植实验我们还发现抑制sFBP的翻译后，神经嵴细胞的正常分化和迁移都受到抑制。但是，显微注射叶酸及其类似物或者显微注射甲基供体S-腺苷甲硫氨酸或者亮氨酸甲基转移酶都不能挽救阻断sFBP造成的表形，由此提示sFBP可能不是通过叶酸传统的参与营养合成或者甲基化的途径发挥作用。我们发现注射sFBP morpholino可以抑制Islet-1mRNA和蛋白质的表达，Islet-1的表达区域与sFBP类似。共同注射Islet-1 mRNA和sFBP morpholino可以极大的挽救sFBP morpholino的表型。最后通过morpholino特异阻断Islet-1的表达后，我们发现其表现出与sFBP morpholino类似的粘液腺发育缺陷，神经板细胞凋亡，小头无眼的表形。由此我们认为sFBP结合叶酸后可能通过细胞膜上的受体传递信号，并且Islet-1可能在sFBP的下游发挥作用。 神经嵴是脊椎动物特有的一群多潜能干细胞，产生于表皮和神经板的边界，在原肠运动之后这群细胞通过表皮间充值转换从神经管背侧迁移到不同的区域，分化成不同的细胞类型，包括外周神经系统，色素细胞，软骨等。神经嵴的发生是一个多步骤多基因参与的精细调控过程。目前理论认为最初由一些分泌性信号分子又叫形态生成素比如BMP,Wnt，FGF，Notch等通过不同浓度梯度的相互作用调节一组在表皮和神经板边界的转录因子（Msx、Pax3/7、Zic1、Dlx3/5等）的表达，即边界决定。这些边界决定因子进一步在预定形成神经嵴的区域激活神经嵴特化基因比如Slug/Snail、FoxD3、Twist、Sox9/10的表达完成神经嵴的特化（Specification）。 Nkx6.3是Nkx6家族的一个转录因子，RT-PCR显示其呈现母源性表达。特异抗体显示Nkx6.3蛋白第9期在整个胚胎都表达，大部分蛋白集中在细胞核，有少部分蛋白定位于细胞膜上；神经板时期主要定位于神经嵴区域的细胞膜上。过表达Nkx6.3会影响细胞粘连分子的表达，由此干扰正常的胚胎原肠运动和Activin诱导的动物帽聚集延伸运动。显微注射Nkx6.3特异morpholino阻断其蛋白表达会抑制神经嵴的marker基因Wnt8，Fgf8，Pax3，Msx1，Zic1，FoxD3，Slug的转录，阻碍神经嵴的发育。在动物帽中单独注射Nkx6.3可以在mRNA水平上诱导Wnt8、Fgf8另一方面抑制BMP4的表达进而诱导神经嵴基因Pax3，Zic1，Slug的表达。报告基因实验也显示Nkx6.3能够激活Wnt信号而在动物帽中抑制BMP信号。Nkx6.3蛋白功能域分析发现其EH1结构域（domain）参与对Wnt8信号的激活，而EH1结构域和HD结构域之间的连接区域（linker domain）参与对FGF的激活和对BMP的抑制。进一步在动物帽和胚胎中分析发现Nkx6.3对Wnt8的激活依赖于FGF家族受体信号但是不依赖于Fgf8。有趣的是4细胞时期过表达Nkx6.3促进Fgf8和Wnt8 mRNA表达，但是抑制边界决定基因Msx1、Pax3和神经嵴特化基因Slug的转录。在32细胞时期显微注射Nkx6.3可以在内源神经嵴发生区域抑制Slug的表达，而异位却诱导Slug的mRNA。我们发现与动物帽中对BMP的调节不同，在胚胎中，过表达Nkx6.3会强烈的激活Smad1蛋白在细胞核中的表达即BMP信号被激活，高的BMP信号会抑制神经嵴的发生。另外我们发现过表达Nkx6.3在胚胎中抑制Dlx5而在动物帽中却不影响Dlx5的表达水平，Morpholino阻断Dlx5会抑制Msx1、Pax3和Slug的表达。BMP信号和Dlx5在动物帽和在整体胚胎中对Nkx6.3的不同响应可以一定程度上解释过表达Nkx6.3在2个系统中对神经嵴基因Slug相反的影响结果。

与黑素生成相关的生化指标测定为主的美白评价体系的初建

近年来，随着黑素细胞生物学的发展和对美白剂功效研究的不断深入，美白剂对皮肤正常生理潜在的负面影响正逐渐为人们所重视 ，仅仅通过临床皮肤敏感试验已远不能给以科学的解释，因而建立一个科学量化的美白剂评价体系就显得尤为迫切和必要。本文工 作旨在将体外黑素细胞原代培养与黑素生成相关的生化指标（包括：黑度，细胞量，铬氨酸酶TYR，多巴色素异构酶DT酶活的）， 检测相结合，并联系细胞学观察，从而形成一整套相对完善，定性定量的研究美白剂功效的评价体系。通过测定外加a-MSH,内皮素 ET-1及其拮抗剂GD2168后黑素细胞内上述生化指标的变化，不仅验证了a-MSH，内皮素ET-1的促黑作用，内皮素拮抗剂GD2168的美 白功效，而且还体现出本评价体系不同于国内现有的其它美白评价体系的独特优势。首先，本评价体系在国内为首家将原代培养的 黑素细胞用于美白评价，由于原代培养环境与体内皮肤生理环境的相似性，在体外对10-8mol/La-MSH产生了正常的应答，所以在评 价美白剂的功效时该培养体较之将黑素细胞孤立生长的纯化培养体系更为科学也更具有说服力。尤其值得一提的是，国内现有的生 化水平的美白评价多局限于对TYR活力的测定，而本体系的另一特点就是除了TYR外还增加了对TRP-2即DT酶活的测定，由于DT在维 持正常黑素细胞及皮肤生理上的重要意义，对其变化的研究往往可揭示出美白剂对皮肤的潜在毒性，本文通过测定并分析内皮素拮 抗剂GD2168对体外黑素细胞DT活力的下调作用，对其潜在的副作用进行了科学的预测，这项工作国内尚无人开展。Over the past few years, melanin cell research has experienced unprecedent impetus, which also contribute to the study on lightener's function especially it's potent skin damage. As a result, it's the high time to build a more accurate and complete evaluation system to investigate lightener's effects and by-effects as well. After normal human melanocytes were cultured primarily in vitro, the effects of a-MSH, endothelin-1(ET-1)and ET -1's antagonist GD2168 on melanogenesis were studied biochemically by measurement of melanin content, cell-number, tyrosinaseTYR activity and dopachrome tautomeraseDT activity. Compared with untreated cells, the treated cells responsed to 10-8mol/L a-MSH with the increase in all items. ET-1 induced both an increae in DT activity and melanin concent; however, the melanosynthesis increase was inhibited significantly in the present of GD2168. Trough above work, a new evaluation system of lightener has been established and confirmed to be feasible. Different from other evaluation systems present in country, this system used the primary culture, which was more consistent to the physiological circumstance. Moreover, the system added DT activity assay that help reveal the GD2168's potent side-effects, which would have been clouded or ignored if only TYR activity was assayed.