Molecular cloning and functional analysis of cathepsin B in nutrient metabolism during larval development in Meretrix meretrix

Seed rearing is an important part in large scale clam culture industry. Since the nutritional history affects early development in bivalve, the condition of larval nutrition plays a key role in successful seed rearing. So far, the molecular mechanism of nutrient uptake in bivalve larvae is unclear. As one of the important proteolytic enzymes, cathepsin B of several organisms has been reported to be involved in digestion. We intended to analyze whether cathepsin B is involved in larval nutrient metabolism in the economic bivalve, clam Meretrix meretrix. The full length of M. meretrix cathepsin B (MmeCB) cDNA was cloned, which is 1647 bp with an open reading frame of 1014 bp. The deduced amino acid sequence encoded a preproenzyme of 337 residues with Cys-114, His-282 and Asn-302 composing cathepsin B activity center. The temporal and spatial expressions of MmeCB mRNA were examined from trochophore to post larva stages by whole mount in situ hybridization. In trochophore stage, no detectable signal was found. In the later three stages, MmeCB mRNA was detected in the digestive gland, suggesting a possible role of MmeCB in digestion. Moreover, MmeCB mRNA was also observed in the epidermal cells in D-veligers. Cathepsin B specific inhibitor (CA074 methyl ester) was applied to block the activity of cathepsin B in unfed larvae. The average shell lengths of treated larvae were smaller than that in control groups. The results of mRNA epidermal distribution and inhibitor treatment in D-veligers indicated that MmeCB may be also associated with other pathway of nutrient metabolism in larval epidermis. The overall results in this paper revealed that MmeCB might play a role in larval nutrient metabolism. (C) 2008 Elsevier B.V. All rights reserved.

Molecular cloning and responsive expression to injury stimulus of a defender against cell death 1 (DAD1) gene from bay scallops Argopecten irradians

Apoptosis is an active process of cell death, which is an integral part of growth and development in multicellular organisms. The defender against cell death 1 (DAD1), the regulatory protein to inhibit the apoptosis process, was first cloned from the bay scallop Argopecten irradians by randomly sequencing a whole tissue cDNA library and rapid amplification of cDNA end (RACE). The full-length cDNA of the A. irradians DAD1 was 607 bp, consist of a 5'-terminal untranslated region (UTR) of 63 bp, a 3'-terminal UTR of 205 bp with a canonical polyadenylation signal sequence AATAAA and a poly (A) tail, and an open reading frame of 339 bp. The deduced amino acid sequence of the A. irradians DAD1 showed 75.5% identity to Araneus ventricosus, 74.5% to Drosophila melanogaster, and 73.6% to Homo sapiens, Sus scrofa, Mesocricetus auratus, Rattus norvegicus and Mus musculus. Excluding the Saccharomyces cerevisiae DAD1 homologue, all animal DAD1 including A. irradians DAD1 homologue formed a subgroup and all plant DAD1 proteins formed another subgroup in the phylogenetic analysis. The A. irradians DAD1 was expressed in all examined tissues including adductor muscle, mantle, gills, digestive gland, gonad and hemolymph, suggesting that A. irradians DAD1 is expressed in most body tissues. Furthermore, the mRNA expression levels of A. irradians DAD1 gene of hemolymph were particularly high after injury, suggesting that the gene is responsive to injury stimuli.

对虾性别分化、性腺发育中若干关键问题的研究

利用扫描电镜和组织学研究相结合的方法研究了中国对虾早期的性别分化。扫描电镜观察的结果表明，中国对虾外部性征的性别分化是在变为仔是后第35天开始的，而内部性征则是在变为仔虾后的第76天观察到生殖腺原基向精巢和卵巢的分化。促雄腺(Androgenic gland)是控制甲壳动物性别分化的一个重要器官。到目前为止，已在软甲类的多数目中报道了促雄腺的存在。不同种类促雄腺的分布位置不同，外部形态也有很大差别。中国对虾的促雄腺位于第五对步足基部的肌肉间，于片状覆盖在精荚囊与射精管连接处。当用苏木精染色时，由于其嗜碱性比较强，整个腺体被染成深蓝色。扫描电镜观察可见，中国对虾的促雄腺具有葡萄串状的外形，它是由许多葡萄形的亚单位组成的，这些亚单位的大小、形态各异组织学观察的结果表明，中国对虾的促雄腺由两种类型的细胞组成。第一类细胞，数量少，核的嗜碱性很强，被苏木精染成深蓝色，核仁不易分辨；第二类细胞，数量大，核比较膨大，嗜碱性较弱，核质疏松，具有明显可见的核仁。不同大小的虾中，腺细胞的排列方式不同。随着对虾的发育成熟，腺细胞逐渐由索状排列变为成团排列，说明促雄腺的形成存在一个逐渐发育的过程。利用手术破坏促雄腺或用促雄腺匀浆液浸泡仔虾的方法研究了中国对虾促雄腺的功能，中国对虾促雄腺的存在与否对于雄性外部性征的发育和维持起着重要作用。破坏促雄腺的雄虾的交接器的发育明显受到抑制。但是，未观察到促雄腺对对虾内部性征发育的影响，这可能与进行手术时对虾所处的发育期有关。利用促雄腺的匀浆液浸泡仔虾，对雄虾交接器的发育有一定促进作用。高温处理对中国对虾受精卵孵化率的影响随处理温度及处理时间的改变而发生变化。用热处理方法成功地诱导了中国对虾三倍体，最高诱导率达75%，并对三倍体群体的生物学特性进行了研究。在中国对虾三倍体的群体中，雌体的巢存在两种类型：一种类型卵巢，生殖细胞停留在卵原细胞阶段，没有进一步发育；另一种类型的卵巢，生殖细胞由卵原细胞发育为初级卵母细胞，发育情况与对虾的类似。对卵巢处于发育的第II或第III期的中国对虾以及卵巢发育处在第I、II、III期的几纳对虾的血液和卵巢中法呢烯酸甲酯（MF）含量进行了研究，结果表明，中国对虾和凡纳对虾中均存在MF。凡纳对虾血液和卵巢发育具有明显的刺激作用，这种刺激作用是通过血液中法呢烯酸甲酯含量的提高来实现的。这一研究结果进一步说明了法呢烯酸甲酯与甲壳动物繁殖之间的关系。

Toxin-screening and identification of bacteria isolated from highly toxic marine gastropod Nassarius semiplicatus

Bacteria isolated from a highly toxic sample of gastropod Nassarius semiplicatus in Lianyungang, Jiangsu Province in July 2007, were studied to probe into the relationship between bacteria and toxicity of nassariid gastropod. The toxicity of the gastropod sample was 2 x 10(2) mouse unit (MU) Per gram Of tissue (wet weight). High concentration of tetrodotoxin (TTX) and its analogues (TTXs) were found in the digestive gland and muscle of the gastropod, using high performance liquid chromatography coupled with mass chromatography (LC-MS). Bacterial strains isolated from the digestive gland were cultured and screened for TTX with a competitive ELISA method. Tetrodotoxin was detected in a proportion of bacterial strains, but the toxin content was low. Partial 16S ribosomal DNA (rDNA) of the TTX-producing strains was then sequenced and compared with those published in the GenBank to tentatively identify the toxic strains. It was found that most of the toxic strains were closely affiliated with genus Vibrio, and the others were related to genus Shewanella, Marinomonas, Tenacibaculum and Aeromonas. These findings suggest that tetrodotoxin-producing bacteria might play an important role in tetrodotoxin accumulation/production in N. semiplicatus. (C) 2008 Elsevier Ltd. All rights reserved.

Identification of quantitative trait loci for growth-related traits in the Pacific abalone Haliotis discus hannai Ino

The locations and effects of quantitative trait loci (QTL) were estimated for nine characters for growth-related traits in the Pacific abalone (Haliotis discus hannai Ino) using a randomly amplified polymorphic DNA (RAPD), amplification fragment length polymorphism (AFLP) and SSR genetic linkage map. Twenty-eight putatively significant QTLs (LOD > 2.4) were detected for nine traits (shell length, shell width, total weight, shell weight, weight of soft part, muscle weight, gonad and digestive gland weight, mantle weight and gill weight). The percentage of phenotypic variation explained by a single QTL ranged from 8.0% to 35.9%. The significant correlations (P < 0.001) were found among all the growth-related traits, and Pearson's correlation coefficients were more than 0.81. For the female map, the QTL for growth were concentrated on groups 1 and 4 linkage maps. On the male map, the QTL that influenced growth-related traits gathered on the groups 1 and 9 linkage maps. Genetic linkage map construction and QTL analysis for growth-related traits are the basis for the marker-assisted selection and will eventually improve production and quality of the Pacific abalone.

LC-DAD-ESI-MS Characterization of Carbohydrates Using a New Labeling Reagent

A novel labeling reagent 1-(2-naphthyl)-3-methyl-5-pyrazolone (NMP) coupling to liquid chromatography with electrospray ionization mass spectrometry for the detection of carbohydrates from the derivatized rape bee pollen samples is reported. Carbohydrates are derivatized to their bis-NMP-labeled derivatives. Derivatives showed an intense protonated molecular ion at m/z [M+H](+) in positive-ion detection mode. The mass-to-charge ratios of characteristic fragment ions at m/z 473.0 could be used for the accurately qualitative analysis of carbohydrates. This characteristic fragment ion is from the cleavage of C2-C3 bond in carbohydrate chain giving the specific fragment ions at m/z [MH-CmH2m+1Om-H2O](+) for pentose, hexose and glyceraldehydes and at m/z [MH-CmH2m-1Om+1-H2O](+) for alduronic acids such as galacturonic acid and glucuronic acid (m = n - 2, n is carbon number of carbohydrate). No interferences for all aliphatic and aromatic aldehydes presented in natural environmental samples were observed due to the highly specific parent mass-to-charge ratio and the characteristic fragment ions. The method, in conjunction with a gradient elution, offered a baseline resolution of carbohydrate derivatives on a reversed-phase Hypersil ODS-2 column. The carbohydrates such as mannose, galacturonic acid, glucuronic acid, rhamnose, glucose, galactose, xylose, arabinose and fucose can successfully be detected.

Application of 2-(11H-benzo[a] carbazol-11-yl) Ethyl Carbonochloridate as a Pro-column Derivatization Reagent of Amino Acid by High Performance Liquid Chromatography with Fluorescence Detection

On a reversed phase Hypersil BDS C-18 (200 mm x 4. 6 mm, 5 mu m) column, 20 amino acids, which were derivatized using 2-(11H-benzo [a] carbazol-11-yl) ethyl carbonochloridate (BCEC-Cl) as pre-column derivatization reagent, were separated in conjunction with a gradient elution. Optimum derivatization was obtained by reacting of amino acids with BCEC-Cl at room temperature for 5 min in the presence of sodium borate catalyst in acetonitrile solvent. The fluorescence excitation and emission wavelengths were 279 nm and 380 nm respectively. The identification of amino acid derivatives from hydrolyzed bovine serum albumin and bee pollen was carried out by post-column mass spectrometry with electrospray ion source in positive ion mode. Linear correlation coefficients of the amino acid derivatives were > 0.9990, and detection limits (at signal to noise of 3:1) were 1.49 - 19.74 fmol for the labeled amino acids.

Detection of carbohydrates using new labeling reagent 1-(2-naphthyl)-3-methyl-5-pyrazolone by capillary zone electrophoresis with absorbance (UV)

A novel labeling reagent 1-(2-naphthyl)-3-methyl-5-pyrazolone (NMP) coupled with capillary electrophoresis (CE) with DAD detection for the determination of carbohydrates has been developed. The chromophore in the 1-phenyl-3-methyl-5-pyrazolone (PMP) reagent is replaced by naphthyl functional group, which results in a reagent with very high molar absorptivity (epsilon(251nm) = 5.58 x 10(4) L mol(-1) cm(-1)). This pen-nits NMP-labeled carbohydrates to be detected with UV absorbance in standard 50-mu m-i.d. fused silica capillaries by zone electrophoresis. in this mode, nanomolar concentrations of detection limits are obtained. The method for the derivatization. of carbohydrates with NMP is simplified. The derivatization reaction is rapid and mild in the presence of ammonia catalyst without further transfer steps. Nine monosaccharide derivatives such as mannose, galacturonic acid, glucuronic acid, rhamnose, glucose, galactose, xylose, arabinose and fucose can successfully be detected in CE mode. Good reproducibility can be obtained with relative standard deviation (R.S.D.) values of the migration times and peak area, respectively, from 0.44 to 0.48 and from 3.2 to 4.8. Furthermore, the developed method has been successfully applied to the analysis of carbohydrates in the hydrolyzed rape bee pollen samples. (C) 2008 Published by Elsevier B.V.

Analysis of saturated free fatty acids from pollen by HPLC with fluorescence detection

A sensitive method for the determination of free fatty acids using 2-(2-(anthracen-10-yl)-1H-naphtho[2,3-dimidazol-1-yl) ethyl-p-toluenesuIfonate (ANITS) as tagging reagent with fluorescence detection has been developed. ANITS could easily and quickly label fatty acids in the presence of the K2CO3 catalyst at 90 degrees C for 40 min in N,N-dimethylformamide solvent. From the extracts of rape bee pollen samples, 20 free fatty acids were sensitively determined. Fatty acid derivatives were separated on a reversed-phase Eclipse XDB-C8 column by HPLC in conjunction with gradient elution. The corresponding derivatives were identified by post-column APCI/MS in positive-ion detection mode. ANITS-fatty acid derivatives gave an intense molecular ion peak at mlz [M+H](+); with MS/MS analysis, the collision-induced dissociation spectra of m/z [M+H](+) produced the specific fragment ions at mlz [M-345](+) and mlz 345.0 (here, m/z 345 is the core structural moiety of the ANITS molecule). The fluorescence excitation and emission wavelengths of the derivatives were lambda(ex) = 250 nm and lambda(em) = 512 nm, respectively. Linear correlation coefficients for all fatty acid derivatives are > 0.9999. Detection limits, at a signal-to-noise ratio of 3 : 1, are 24.76-98.79 fmol for the labeled fatty acids.

A preliminary analysis of the phylogeny of the Swertiinae (Gentianaceae) based on ITS data

To better understand the floral diversity and the phylogeny of the Swertiinae (Gentianaceae), the internal transcribed spacer (ITS) region of nrDNA for 17 species and one outgroup was sequenced. Our data suggest that corolla type, gland shape, and corolla appendage are poorly correlated with the ITS phylogeny. The genus Swertia s.l. and the rotate group previously recognized based on the corolla types and gland shapes are polyphyletic. Four genera with simple protruding glands and three taxa with corolla appendages are not clustered as the monophyletic groups. Four separate clades, corresponding to the four sections, were identified in Swertia s.1. Lomatogoniopsis with the simple protruding gland type of the tubular group closely related to Lomatogonium of the rotate group. The deeply lobing corolla and concave foveae may be ancestral in the Swertiinae, while the tubular corolla and the protruding glands may have undergone convergent evolution.

RFID系统优化模型与智能算法研究

射频识别技术（Radio Frequency Identification, RFID）作为采集与处理信息的高新技术和信息化标准的基础，被列为本世纪十大重要技术之一。但是，RFID技术的大规模实际应用仍处于探索阶段，RFID系统的应用基础技术还存在着大量尚未解决的关键问题，其中RFID系统优化是RFID技术研究和应用的重要课题。由于RFID系统本身的动态性和不确定性， RFID系统优化面对的一般是非线性、多目标、大规模的复杂优化问题，传统的数学优化算法在处理这些问题时，存在困难。为此，研究新的优化算法成为RFID技术实际应用和理论研究中必须解决的课题。 智能计算方法是求解复杂RFID系统优化问题的一种可供选择的算法。智能计算作为一个新兴领域，其发展已引起了多个学科领域研究人员的关注，目前已经成为人工智能、经济、社会、生物等交叉学科的研究热点和前沿领域。智能计算的各类算法已在传统NP问题求解及诸多实际应用领域中展现出其优异的性能和巨大的发展潜力。 本文旨在对RFID系统的各种优化问题进行深入研究和探讨，面向RFID技术的实际应用需求构建其优化模型，并基于智能计算思想设计能够有效求解这些复杂模型的新型智能优化算法。具体研究内容包括： 首先，进行了RFID读写器网络的调度问题研究。在深入分析RFID网络中读写器冲突类型和成因的基础上，考虑RFID网络中的读写器冲突约束，以最小化系统中的频道数量、时隙分配以及总处理时间建立了RFID读写器网络调度的数学优化模型。从生物学的角度出发提出基于生态捕食模型的改进PSO算法（Particle Swarm Optimizer based on Predator-prey Coevolution, PSOPC），在一定程度上解决了PSO算法在迭代后期随着多样性丧失而陷入局部最优的缺点。应用PSOPC设计了求解RFID读写器网络调度模型的智能求解算法，分别给出算法的求解框架、关键步骤的实现机制。通过在不同规模的RFID读写器网络上进行实例仿真，验证了算法的有效性和模型的正确性。 其次，进行了基于菌群自适应觅食算法RFID网络规划问题的研究。考虑RFID系统在不同应用环境下的系统需求，建立了RFID网络规化的数学模型，其目标函数分别为：RFID网络标签覆盖率的最大化目标函数、RFID读写器冲突的最小化目标函数、RFID网络运行的经济效益最大化目标函数、RFID网络运行的负载平衡目标函数以及同时考虑全局目标的混合目标函数。将自然界生物觅食所采用的自适应搜索策略与细菌的趋化行为和群体感应机制相集成，提出了适合求解复杂RFID网络规划问题的菌群自适应觅食算法（Adaptive Bacterial Foraging Optimization, ABFO）。通过仿真实验基于ABFO算法分别对RFID网络规划模型中的五个目标函数进行了实例求解和分析，测试结果与标准PSO算法和遗传算法进行了比较分析。 再次，进行了基于系统智能方法的RFID网络规划分布式决策模型研究。采用分布式决策的思想建立了RFID网络规划的层次模型，在一定程度上缓解、分散了RFID网络规划问题的复杂性，以解决具有混合变量（包括离散变量和连续变量）的多目标RFID网络规划问题。针对层次模型求解的复杂性，以复杂适应系统理论为指导思想设计了一种新型系统智能优化算法对RFID网络规划的层次模型进行求解。系统智能算法将群体智能中的单层群体系统概念扩展为多层涌现系统，仿真实验表明新提出的算法显著提高了智能计算方法的寻优能力，以及算法的适应性、鲁棒性和平衡性等性能。 最后，进行了RFID网络目标跟踪系统中的数据融合研究。以基于RFID技术的目标定位与跟踪系统为应用背景，提出了基于模糊聚类方法的多RFID读写器数据融合模型框架。通过深入分析蜜蜂采蜜的基本生物学规律，对蜜蜂的个体行为及群体行为进行模拟，提出了一类新型群体智能优化算法-蜂群优化算法（Bee Swarm Optimization, BSO），并将BSO算法嵌入RFID目标定位跟踪系统，作为其模糊聚类的基本算法。仿真研究表明，提出的融合模型能够有效的过滤读写器对跟踪目标的错误监测数据，显著提高目标定位与跟踪的精度。

800MPa下泥质岩部分熔融实验研究：残留相及熔体相REE地球化学特征

利用JL-3600t压机实验研究了800MPa、不同温度条件下泥质岩部分熔融过程，利用EMPA和LA-ICPMS分别测定了熔体相和残留相中主要化学组成以及微量元素（包括REE）组成。实验结果表明，泥质岩低程度部分熔融（〈25％）形成的熔体中REE含量分布于308．8-3565μg／g较大范围内，显示较大的不均匀性，其REE球粒陨石标准化分布模式显示弱的M型REE“四分组效应”，而残留相矿物石榴子石中REE含量变化于167．5—1008μg／g范围，也显示有明显的不均匀性，其REE球粒陨石标准化分布模式显示明显的W型REE“四分组效应”，尤以第一段La-Nd最为显著；随着部分熔融程度的增加（〉30％），其形成的熔体中REE集中在523．2—1130μg／g范围，残留相石榴子石中BEE集中在288．6—512．7μg／g范围，均显示相对均匀；熔体相和残留相石榴子石矿物的REE球粒陨石标准化分布模式不发育REE“四分组效应”。实验前后Cl质量平衡计算的结果表明该实验过程中并没有产生岩浆挥发分相。上述特征表明S型花岗岩中的REE“四分组效应”现象很可能与泥质岩低程度部分熔融具有成因联系。

生物钟基因mPer2调控节律性免疫功能的机制研究

This project was carried out with the aims to investigate the mechanism of circadian immune regulation by one of the core Clock gene, mPer2. To achieve this, we selected mPer2 knock out (mPer2-/-) mice as the optimal animal model. Two different approaches were performed. In the first approach, we injected WT or mPer2-/- mice with an equal dosage of lipopolysaccharide (LPS), and systematically measured serum corticosterone induction, expression of core Clock genes, as well as a key enzyme for corticosterone metabolism (mStAR) in adrenal gland. We found that the acute induction of corticosterone and mStAR were closely associated with the circadian immune response to LPS. Besides, real time quantitative PCR (q-PCR) and luciferase assay consistently showed that mStAR is a Clock controlled gene in adrenal gland, where its expression is negatively influenced by mPer2. In the second approach, expression level and circadian manner of 11 cytotoxicity regulation genes in WT or mPer2-/- mice bone marrow were measured by q-PCR in order to explore the candidate genes which could mediate the circadian immune regulation by mPer2. We found that expression level of Ly49C, Ly49I, and Nkg2d was significant down-regulated in mPer2-/- mice. Further, we found that daily expression of Ly49C and Nkg2d fluctuated in a circadian manner in WT mice, where these rhythms were disrupted in mPer2-/- mice. Thus, it was suggested that these two cytotoxic genes were two clock controlled genes whose circadian expression were regulated by mPer2. Taken together, our results suggested that corticosterone, mStAR, Ly49C, and Nkg2d were four candidate molecules that may mediate the circadian immune response regulation by mPer2.

Pancreatic and adrenal development and function in an ovine model of polycystic ovary syndrome.

Polycystic Ovary Syndrome (PCOS) is a complex disorder encompassing reproductive and metabolic dysfunction. Ovarian hyperandrogenism is an endocrine hallmark of human PCOS. In animal models, PCOS-like abnormalities can be recreated by in utero over-exposure to androgenic steroid hormones. This thesis investigated pancreatic and adrenal development and function in a unique model of PCOS. Fetal sheep were directly exposed (day 62 and day 82 of gestation) to steroidal excesses - androgen excess (testosterone propionate - TP), estrogen excess (diethylstilbestrol - DES) or glucocorticoid excess (dexamethasone - DEX). At d90 gestation there was elevated expression of genes involved in β- cell development and function: PDX-1 (P<0.001), and INS (P<0.05), INSR (P<0.05) driven by androgenic excess only in the female fetal pancreas. β- cell numbers (P<0.001) and in vitro insulin secretion (P<0.05) were also elevated in androgen exposed female fetuses. There was a significant increase in insulin secreting β-cell numbers (P<0.001) and in vivo insulin secretion (glucose stimulated) (P<0.01) in adult female offspring, specifically associated with prenatal androgen excess. At d90 gestation, female fetal adrenal gene expression was perturbed by fetal estrogenic exposure. Male fetal adrenal gene expression was altered more dramatically by fetal glucocorticoid exposure. In female adult offspring from androgen exposed pregnancies there was increased adrenal steroidogenic gene expression and in vivo testosterone secretion (P<0.01). This highlights that the adrenal glands may contribute towards excess androgen secretion in PCOS, but such effects might be secondary to other metabolic alterations driven by prenatal androgen exposure, such as excess insulin secretion Thus there may be dialogue between the pancreas and adrenal gland, programmed during early life, with implications for adult health Given both hyperinsulinaemia and hyperandrogenism are common features in PCOS, we suggest that their origins may be at least partially due to altered fetal steroidal environments, specifically excess androgenic stimulation